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Bovine neosporosis: From life cycle to prophylaxis
Abstract
Neospora caninum is an apicomplexan parasite, identified about twenty years ago. Although it can cause neurological symptoms in newborn cattle, its main economic and health repercussion in breeding is due to abortions. The life cycle of N. caninum is not yet fully understood. Its currently identified final hosts are dogs and coyotes, but foxes are also strongly suspected. To be effective, treatment and prophylaxis must be based on sound knowledge of the life cycle and pathophysiology of this protozoosis. There is no treatment currently available. Many vaccine studies are being performed using various strategies, but only disease control methods are possible for the moment, alongside the crucial advisory role of veterinary practitioners.
Neospora caninum is currently considered as one of the major pathogens responsible for abortions in cattle, which acts as an intermediate host. Dogs are the definitive hosts, but they also act as intermediate hosts for this parasite. This raises the legitimate question as to whether N. caninum is indeed responsible for reproductive disorders in dogs, given that the worldwide seroprevalence of the infection is far fromnegligible among the canine populations. There are arguments in favour of an adverse effect of N. caninum during pregnancy in bitches: firstly, the vertical transmission of this parasite in dogs is practically proven, and secondly, experimental studies with the inoculation of pregnant bitches induced foetal or neonatal infections. However, results are contradictory and more systematic and rigorous studies must be carried out before any conclusion can be drawn on the prevalence of this parasite in reproductive disorders in pregnant bitches.
Der einzellige Parasit Neospora caninum gilt weltweit als einer der wichtigsten infektiosen Abortverursacher beim Rind. In einer schweizerischen Fall-Kontroll-Studie wurden 113 Abortproblem- und 113 Kontrollbetriebe untersucht. Dabei konnte mittels PCR N. caninum im Hirn von 21% der insgesamt 242 untersuchten abortierten Feten nachgewiesen werden. Die zu einem N. caninum-Abort gehorenden Muttertiere waren zu 84% N. caninum-seropositiv. Betreffend aller untersuchten 226 abortierenden Muttertiere ergab sich eine N. caninum-Seropravalenz von 44%. Seroepidemiologisch wurden 4505 Rinder im Zeitabstand von 3–12 Monate 2-mal serologisch untersucht. Dabei traten temporal starke Schwankungen in der Antikorperkonzentration auf, so dass 39% der vormals positiven Tiere in der zweiten Untersuchung keine nachweisbaren Antikorper mehr gegen N. caninum aufwiesen. Eine retrospektiv durchgefuhrte Befragung von 42 Problem- und 42 Kontrollbetrieben zeigte, dass sich in mehr als 80% der vormaligen Problembetriebe die Abortsit...
C57BL/6 mice were infected with Neospora caninum tachyzoites during pregnancy, yielding a transplacental infection of developing fetuses. Subsequently, congenitally infected newborn mice were treated either once or three times with toltrazuril (or placebo) at a concentration of 31.25 mg compound per kg body weight. Both toltrazuril and placebo treatment had no negative effect on newborns, as noninfected treated pups developed normally without differences in mortality and morbidity to matching nontreated control animals. Already one application of toltrazuril was significantly (p < 0.01) able to delay the outbreak of neosporosis in newborn mice, when compared to placebo-treated infected controls. We found significantly higher proportion of surviving newborns in one-time-toltrazuril-treated and three-time-toltrazuril-treated groups (34% and 54%, respectively) when compared to one-time-placebo-treated and three-time-placebo-treated groups (14% and 30%, respectively). There was no significant difference (p = 0.2) in the proportion of surviving pups between one-time-toltrazuril and three-time-toltrazuril treatment. However, the number of diseased and Neospora-positive pups (46% and 47%, respectively) was markedly reduced after three-time-toltrazuril treatment compared to all other groups. Three-time-treatment also resulted in the highest antibody (IgG, IgG2a) response. Pharmacokinetic analyses using individual serum samples revealed that, although toltrazuril was absorbed and metabolized to toltrazuril sulfone by newborn mice, medicated animals exhibited an unexpected rapid turn-over (half-life time) of the compound. Toltrazuril and the metabolite were also found in brain tissues, indicating that passage of the blood-brain barrier occurred. In conclusion, we could show that three times treatment with toltrazuril had a high impact on the course of infection in congenitally N. caninum-infected newborn mice.
Tachyzoites of 2 isolates of Neospora caninum (NC-1 and NC-2) were inoculated subcutaneously (s.c.), intraperitoneally (i.p.), or orally into mice to compare the effects of route of inoculation on pathogenicity. Mice developed more severe disease, and disease occurred sooner when inoculated with the NC-1 isolate compared to the NC-2 isolate. Deaths occurred earlier in mice inoculated i.p. with either isolate. Mice inoculated orally or s.c. with tachyzoites responded similarly to infection. Tissue cysts of the NC-2 isolate produced infections in mice following oral or s.c. inoculation. Lesions seen in mice inoculated with tachyzoites or bradyzoites were primarily acute pneumonia, myositis, encephalitis, ganglioradiculoneuritis, and pancreatitis. In vitro studies demonstrated that tachyzoites of both isolates were killed by incubation in pepsin-HCl solution but not 1% trypsin solution. Bradyzoites of the NC-2 isolate were able to withstand treatment with pepsin-HCl solution.
Neospora caninum causes serious disease in dogs, and it, or a similar parasite, is a major cause of abortion in cattle. Little is known about the susceptibility of this protozoan to antimicrobial agents. We studied several antimicrobial agents to determine which classes might have activity against this parasite. We also determined whether activity of such agents was coccidiocidal or coccidiostatic. A 2-day of treatment, monoclonal antibody-based enzyme immunoassay and a 5-day of treatment, cell culture flask (CCF), lesion-based assay were developed to examine the ability of test agents to inhibit tachyzoite multiplication. Seven sulfonamides were examined, with the following activities observed: sulfathiazole > or = sulfamethoxazole > sulfadiazine > sulfaquinoxaline > or = sulfamethazine > sulfadimethoxine > sulfamerazine. Dapsone, a sulfone, had little activity. Six dihydrofolate reductase/thymidylate synthase inhibitors were examined, with the following activities observed: piritrexim > pyrimethamine > ormetoprim > trimethoprim = diaveridine > methotrexate. Six ionophorous antibiotics were examined; lasalocid, maduramicin, monensin, narasin, and salinomycin had equivalent activities, but alborixin was toxic for host cells at the lowest concentration examined. Three macrolide antibiotics--azithromycin, clarithromycin, and erythromycin--were examined and had equivalent activities. Two tetracycline antibiotics, doxycycline and minocycline, were examined and had equivalent activities. Three lincosamide antibiotics were examined, with the following activities observed: clindamycin hydrochloride > clindamycin phosphate > lincomycin hydrochloride. Pentamidine and 6 of its analogs were examined, and only hexamidine and 1,4-Di[4-(2-imidazolinyl)-2-methoxy-phenoxy]butane had activity. Eight miscellaneous antiprotozoal agents were examined for activity. Amprolium, metronidazole, paromomycin, and roxarsone had little activity.(ABSTRACT TRUNCATED AT 250 WORDS)
Neospora caninum is a recently recognized protozoan parasite of animals, which until 1988 was misidentified as Toxoplasma gondii. Its life cycle is unknown. Transplacental transmission is the only recognized mode of transmission. It has a wide host range, but its zoonotic potential is unknown. Neosporosis is a major cause of abortion in cattle in many countries. It is also an important cause of neuromuscular paralysis in dogs. This paper reviews information on parasite structure, life cycle, biology, clinical signs, diagnosis, treatment and control.
Dogs were investigated to determine if they are definitive hosts of Neospora caninum. Four dogs were fed N. caninum tissue cysts in infected mouse tissue, and two negative control dogs were fed uninfected mouse tissue. Dog faeces were examined daily for 30 days using a sucrose flotation technique. Three challenged dogs shed spherical to subspherical unsporulated oocysts, measuring 10 to 11 microns in diameter. Oocysts sporulated within 3 days and contained two sporocysts, each with four sporozoites. Outbred, inbred, and gamma-interferon knockout mice were inoculated with canine faecal extracts and monitored for evidence of neosporosis using a variety of morphologic, immunohistologic, serologic, and genetic analyses. Mice that received faeces from each dog observed to shed oocysts were demonstrated to have neosporosis by two or more techniques. One mouse was demonstrated to be infected with N. caninum by immunohistochemistry, ultrastructural analysis, and a species-specific PCR test. No evidence of neosporosis was observed in control animals. Based on this study, dogs are a definitive host of Neospora caninum.
To determine whether cows with evidence of previous infection with Neospora caninum were less likely to abort or give birth prematurely during an outbreak of neosporosis, compared with herdmates with evidence of primary infection.
Cohort study.
208 pregnant beef cows.
Aborted fetuses and calves born prematurely were examined during an outbreak of neosporosis in a herd of beef cows. Sera were collected from all cows during the outbreak and again 71 days later. Cows were classified into groups on the basis of normal and abnormal reproductive outcomes. Sera were examined, using an avidity ELISA procedure for N caninum, and results were compared between groups and among time points.
Antibody concentrations decreased significantly and IgG avidity values increased significantly over time. During the outbreak, cows with normal reproductive outcomes were significantly more likely to have high IgG avidity values than cows with abnormal reproductive outcomes.
The herd had numerous abortions and premature births with evidence of recent point-source exposure to N caninum. Therefore, to reduce risk of transmission of N caninum to cattle, attempts should be made to prevent canine feces from contaminating feed, especially feedstuffs used to prepare mixed rations for cattle. Cows with evidence of previous exposure to N caninum were less likely to abort or give birth prematurely during the outbreak than cows with primary infections with N caninum; this finding suggests development of protective immunity in previously infected cows.
To determine the epidemiologic plausibility of a sylvatic transmission cycle for Neospora caninum between wild canids and beef cattle.
Spatial analysis study.
1,009 weaned beef steers from 94 beef herds in Texas.
Calves were grouped on the basis of seroprevalence for N caninum and ecologic region in Texas. The Morans I test was used to evaluate spatial interdependence for adjusted seroprevalence by ecologic region. Cattle density (Number of cattle/259 km2 [Number of cattle/100 mile2] of each ecologic region) and abundance indices for gray foxes and coyotes (Number of animals/161 spotlight-transect [census] km [Number of animals/100 census miles] of each ecologic region) were used as covariates in spatial regression models, with adjusted seroprevalence as the outcome variable. A geographic information system (GIS) that used similar covariate information for each county was used to validate spatial regression models. Results-Spatial interdependence was not detected for ecologic regions. Three spatial regression models were tested. Each model contained a variable for cattle density for the ecologic regions. Results for the 3 models revealed that seroprevalence was associated with cattle density and abundances of gray foxes, coyotes, or both. Abundances of gray foxes and coyotes were collinear. Results of a GIS-generated model validated these spatial models.
In Texas, beef cattle are at increased risk of exposure to N caninum as a result of the abundance of wild canids and the density of beef cattle. It is plausible that a sylvatic transmission cycle for neosporosis exists.
The baculovirus expression system has proved to be a useful tool for the production of recombinant proteins. Here we have
characterized the Neospora caninum surface protein NcSRS2 produced by two types of the recombinant virus and also have developed an enzyme-linked immunosorbent
assay (ELISA) using recombinant NcSRS2 for the serologic diagnosis ofNeospora infection. Western blot analysis showed two major protein bands that were detectable in insect cells infected with each recombinant
baculovirus, and a lower-molecular-weight protein was detected in culture supernatants from a cell infected with the recombinant
virus lacking the hydrophobic C-terminal tail. Analysis of the N-terminal amino acids showed that the secreted NcSRS2 lacked
6 kDa of the N-terminal signal peptide. Moreover, the detergent-soluble protein of insect cells infected with the recombinant
baculovirus expressing the full-length NcSRS2 gene was used to develop an ELISA system based on specificity and reactivity
to antisera againstToxoplasma gondii, Hammondia heydorni, orN. caninum. Anti-N. caninum mouse, dog, and bovine sera recognized the recombinant NcSRS2 on Western blots. Furthermore, we have shown that the developed
ELISA system consistently discriminates indirect fluorescent-antibody test (IFAT)-positive bovine sera against N. caninum from IFAT-negative sera. These results indicate that the ELISA using baculovirus-expressed NcSRS2 can be useful for effective
and reliable serodiagnosis of N. caninuminfection.
Neosporosis is a disease affecting predominantly fetal development in cattle and dog hosts; and it may cause neuromuscular
disfunction in infected new-born calves and pups. Predispositions – including, e.g. transient immunosuppression during pregnancy
– may result in an increased dissemination of the parasite within the host or its offspring. Chemotherapeutic treatment of
neosporosis may be an issue, provided that an appropriate drug is made available. In this respect, we describe the use of
a mouse model for the evaluation of toltrazuril and ponazuril medication as a means of preventing parasite dissemination and
subsequent formation of cerebral lesions. Toltrazuril- and ponazuril-treated mice were experimentally infected intraperitoneally
(i.p.) with 2 × 106
Neospora caninum tachyzoites. The infection was monitored at three levels: clinically, by assessing symptoms, histologically, by assessing
the occurrence of cerebral lesions and parasites by immunohistochemistry, and on the molecular level, by detection of parasite
DNA using PCR. Chemotherapy using either toltrazuril or ponazuril, both applied in a drinking-water formulation (20 mg toltrazuril
or ponazuril kg−1 body weight day−1) completely prevented the formation of cerebral lesions in all treated animals, as assessed by immunohistochemistry. PCR
analyses of these treated animals showed that DNA-detectability was reduced by 91% and 90% upon toltrazuril and ponazuril
medication, respectively.
Neospora caninum is a major cause of abortion in dairy cattle in the United States and other countries. Abortions and neonatal mortality also occur in other ruminant species. Decoquinate is an anticoccidial that is approved for use in cattle and goats in the United States. We studied the efficacy of decoquinate against tachyzoites of N. caninum in a 5-day of treatment, cell culture flask lesion-based assay. Decoquinate killed tachyzoites at concentrations of 0.1 and 0.01 μg ml−1. Decoquinate had little measurable effect on extracellular tachyzoites. Decoquinate acted quickly to kill intracellular stages at coccidiocidal concentrations; tachyzoites were killed within 5 min at 0.1 μg ml−1 decoquinate.
Dogs from dairy farms with a known prevalence of Neospora caninum antibodies in the cattle were examined for the presence of N. caninum antibodies using an ELISA. Data of farm dogs were compared with those of dogs examined at a university clinic, which originated mainly in urban areas. Of the 152 farm dogs, 36 (23.6%) were seropositive to N. caninum, which was significantly higher than the proportion of seropositives in the clinic dog population (19 of 344, 5.5%). Seroprevalence was significantly higher (P = 0.01) in female dogs than in male dogs. Seroprevalence in dogs increased with age, indicating postnatal infection. Seropositivity to N. caninum in farm dogs was strongly correlated with a high prevalence of N. caninum antibodies in the cattle. At farms where no dogs were present, the seroprevalence to N. caninum in the cattle was significantly lower (P = 0.0002) than in farms where dogs were present. These findings suggest that there is a relationship between N. caninum infection of farm dogs and cattle. Since dogs have been shown to be definitive hosts of N. caninum, cattle may be infected by exposure to canine oocysts. Further research is needed to find out whether and how dogs may acquire the infection from cattle.
A killed whole Neospora caninum tachyzoite preparation was formulated with various adjuvants and tested for its immunogenicity in cattle. The adjuvants used were: Havlogen, a polymer of acrylic acid cross-linked with polyallylsucrose; Polygen, a non-particulate copolymer; a mixture of Havlogen and Bay R-1005, which is a preparation of free base synthetic glycolipids; and Montanide ISA 773, a water-in-oil emulsion made with a mixture of metabolisable and mineral oils. Immune responses in immunised cattle were compared with those of cattle experimentally infected with culture-derived N. caninum tachyzoites. The overall mean serum IFAT titres were significantly higher (P < 0.05) in experimentally infected cattle compared with all immunised cattle. Nonetheless, the maximum antibody titres of the immunised cattle, which were obtained following the third immunisation, were within the range of titres previously described for naturally infected cattle. The overall mean serum IFAT titres were significantly higher (P < 0.05) in cattle immunised with the killed tachyzoite preparation formulated with Polygen and with the mixture of Havlogen and Bay R-1005, compared with cattle immunised with the Havlogen- and Montanide-based preparations. Two of the four adjuvant preparations were able to induce cell-mediated immune responses similar to those of the experimentally infected cattle. The Havlogen-adjuvanted tachyzoite preparation elicited N. caninum-specific proliferation of peripheral blood mononuclear cells statistically similar (P = 0.095) to that of the infected animals. Peripheral blood mononuclear cells from animals immunised with the Polygen-adjuvanted tachyzoite preparation produced interferon-gamma concentrations of similar magnitude (P = 0.17) to those from the infected animals. Polygen was one of two adjuvants that elicited the highest antibody responses, and was the only adjuvant that induced interferon-gamma levels similar to those of the infected heifers.
Dogs are a definitive host of Neospora caninum and cattle are intermediate hosts. Alternative life-cycles have not been investigated. Foxes are frequently seropositive, but may not commonly prey upon cattle; therefore, other intermediate hosts may exist that are frequent prey of foxes. Three domestic pigeons (Columbia livia) and three zebra finches (Poephila guttata) were inoculated with N. caninum tachyzoites, to determine if they could serve as intermediate hosts. Tissue culture, PCR, serology, and histology were all positive for one or more pigeons. All finches resisted infection. Further testing of columbiform birds as intermediate hosts of N. caninum is warranted.
To provide objective data on the potential role of dingoes (Canis lupus dingo) in the life cycle of Neospora caninum in Australia, the production of N. caninum oocysts by experimentally infected canids was investigated. Three dingo pups raised in captivity and three domestic dogs were fed tissue from calves infected with an Australian isolate of N. caninum, Nc-Nowra. Oocysts of N. caninum, confirmed by species-specific PCR, were shed in low numbers by one dingo pup at 12-14 days p.i. The remaining animals did not shed oocysts. Furthermore, the blood from two out of three dingoes tested positive for DNA of N. caninum using PCR tests at 14 and 28 days p.i. Oocyst shedding from the intestinal tract of a dingo demonstrates that dingoes are definitive hosts of N. caninum and horizontal transmission of N. caninum from dingoes to farm animals and wildlife may occur in Australia.
Vertical transmission of the protozoan parasite, Neospora caninum is highly efficient and can take two forms - endogenous transplacental transmission resulting from activation of the quiescent bradyzoite stage during pregnancy or exogenous transplacental transmission resulting from ingestion of oocysts during pregnancy. Calves born carrying infection derived from either endogenous or exogenous transplacental transmission are capable of infecting their offspring when they start to breed. This review considers firstly the frequency with which exogenous and endogenous transmission occur, secondly the role of the immune response in controlling N. caninum infection and thirdly how the parasite persists in an immune-competent host and is re-activated during pregnancy.
Protozoal encephalitic lesions were found in four aborted fetuses and one dead newborn calf. The organism was identified as Neospora caninum by immunoperoxidase. The brain lesions were of two forms. One was observed in three fetuses of 5 months gestation and was characterized by multifocal necrosis. The other was found in a 7-month fetus and in a newborn calf, and showed severe infiltration with macrophages and plasma cells containing IgG. This association, between the age of fetus and inflammation, may reflect development of the immune system in bovine fetuses.
An immunosuppressed mouse model was used to determine the effects of amprolium and sulfadiazine on experimental Neospora caninum infections. Both drugs were given in the drinking water. Neither drug was effective in treating infections when given 7 days after inoculation of tachyzoites, when clinical signs of disease had developed. Amprolium did not prevent deaths or development of clinical signs when given in the drinking water at 1 mg/ml or 5 mg/ml 3 days after inoculation of tachyzoites. Sulfadiazine in drinking water was not effective when given at 0.5 mg/ml but was effective in preventing deaths and clinical disease when given at 1 mg/ml 3 days after inoculation with tachyzoites. Most mice (6 of 10) treated for 3 days with 1 mg/ml sulfadiazine in drinking water developed encephalitis after drug treatment was stopped. Treatment for 14 days with 1 mg/ml sulfadiazine in drinking water was needed to protect 90% of inoculated mice.
Portions of the placenta and kidneys from a 5-month-old bovine fetus were examined histologically. The placental cotyledonary villi were necrotic and protozoa were found in lesions. The organisms were in trophoblasts, periodic acid Schiff-negative and did not react in an immunoperoxidase test using anti-Toxoplasma gondii serum. Individual zoites were approximately 5 x 2 microns and the biggest collection was 45 x 35 microns. The organism in the present case was structurally distinct from Sarcocystis spp. and Toxoplasma gondii, but resembled Neospora caninum.
Neospora caninum tissue cysts were found in sections of the brain from a full-term stillborn deer of Eld (Cervus eldi siamensis) from a zoo in France. There was N. caninum-associated nonsuppurative encephalitis and the diagnosis was confirmed in immunohistochemical staining with antibodies specific to N. caninum.
Neospora caninum is a recently identified coccidian parasite that is closely related to Toxoplasma gondii. Molecules associated with the surface of N. caninum tachyzoites are likely to be involved in the process of adhesion and invasion of host cells. They probably also participate in the interaction of the parasite with the immune system, and they could play an important role in the pathogenesis of the parasite. To identify such surface molecules, we performed subcellular fractionation studies of isolated N. caninum tachyzoites. Employing the nonionic detergent Triton-X-114, we prepared a membrane fraction. Immunoblot analysis of this fraction using polyclonal antisera directed against tachyzoites of N. caninum and T. gondii resulted in the identification of a protein of approximately 43 kDa (Nc-p43). This molecule was present in two isolates of Neospora (Nc-1 and Liverpool) but was absent in Toxoplasma (RH-strain) tachyzoites. Further immunofluorescence and immunogold transmission electron microscopy (TEM) studies using affinity-purified anti-Nc-p43 antibodies demonstrated the presence of this molecule on the surface of N. caninum tachyzoites.
Three groups of eight pregnant sheep were inoculated with tachyzoites of the NCl isolate of Neospora caninum at 45 (group 1), 65 (group 2) or 90 (group 3) days' gestation. A further six animals (group 4) served as controls. Fourteen of the infected ewes developed a fever, which in two cases was biphasic. In six ewes in group 1, the fetuses died and were resorbed, and in the other two the fetuses were aborted. In group 2, one ewe resorbed her fetus, six aborted dead fetuses and one produced a live lamb. In group 3, six ewes aborted and two produced one live and one stillborn lamb each. Thus, the stage of gestation influenced the outcome of infection. All but one of the ewes "seroconverted", as shown by enzyme-linked immunosorbent assay, and 10 of 13 fetal sera examined by an indirect immunofluorescent antibody test were positive. The polymerase chain reaction was also used to detect DNA of N. caninum in aborted tissues. Immunohistochemical examination showed that the parasite had invaded the placentas of all cases examined, displaying an apparent predilection for fetal chorionic epithelium and fetal placental blood vessels, as well as inducing thrombosis in some maternal caruncular blood vessels. Organisms were associated with fetal vasculitis, focal degeneration and inflammation of the chorioallantois, and widespread, severe focal necrosis in the placentome. Characteristic lesions were seen in the fetal brains, in addition to focal leucomalacia, thought to be due to anoxia resulting from the placental damage. The six control sheep in group 4 remained clinically healthy and produced normal uninfected lambs.
Neospora caninum has emerged as a major cause of abortion in cattle in many countries. This paper reviews recent advances in the life cycle and biology of Neospora with the emphasis on bovine neosporosis. The role of the recently discovered oocyst stage of N. caninum in the epidemiology of neosporosis is discussed. Progress made in serologic diagnosis of N. caninum infection is discussed. There is no vaccine for preventing Neospora-induced abortions in cattle or to prevent oocyst shedding in dogs.
Neosporosis is an important cause of abortion and neonatal morbidity in dairy cattle. The disease is caused by Neospora caninum, an intracellular protozoan parasite. In this report, we describe the use of a mouse model in the preliminary evaluation of vaccination as a means to prevent vertical transfer of N. caninum. Parasites present in the tissues of the offspring were detected using an N. caninum-specific polymerase chain reaction assay. Immunization of dams with a single inoculation of a crude lysate of N. caninum tachyzoites appeared to induce complete protection against infection of the offspring.
The protozoan parasite Neospora caninum and bovine virus diarrhoea virus (BVDV) are recognized as important causes of bovine abortion and congenital disease worldwide. In this study, serological investigations were performed to estimate the prevalence of N. caninum infection in Swedish dairy cattle, to assess to what extent it may affect abortion rates, and to determine possible effects of coinfection with BVDV. The overall N. caninum seroprevalence in Swedish dairy cows was estimated at 2% (16/780), and the BVDV seroprevalence was 32% (249/780). Among aborting cows from herds with abortion problems, 7% (26/378) had antibodies to N. caninum and 42% (153/378) to BVDV. Seventeen of the N. caninum positive animals also had antibodies to BVDV. There was a statistically significant (P = 0.013) association between presence of antibodies to N. caninum and BVDV. In a case-control study comprising sera from cows in herds without recognized abortion problems, 6% (5/89) and 1% (1/89) of sera from aborting and non-aborting cows, respectively, had antibodies to N. caninum. Two of the N. caninum seropositive aborting cows also had antibodies to BVDV. These results confirm that N. caninum infection is associated with bovine abortion in Sweden and also indicate that there might be concurrent effects of N. caninum and BVDV. It is concluded that Swedish dairy cows have a low prevalence of N. caninum infection which is favourable in relation to possible future control programmes.
Explosive abortion outbreaks in 4 Dutch dairy herds during 1992 to 1994 are reported. In 50 of 51 fetuses submitted during the first 3 wk of the outbreaks characteristic histological lesions compatible for infection with Neospora caninum were seen. Diagnosis of infection was confirmed by immunohistochemistry in 40 fetuses (78%). No evidence for other abortifacients was found. The abortion risk of the herds was investigated in a prospective and retrospective cohort study. The prospective study showed that cows aborting during the outbreaks and N. caninum seropositive nonaborting cows had a two- to three-fold increased risk of abortion compared with N. caninum seronegative cows. Retrospective examination showed that seropositive nonaborting cows had an increased risk of abortion before the outbreaks, which may indicate that these animals were infected with N. caninum prior to the outbreaks. It is concluded that serostatus can be used for selective culling of cows to decrease future risk of abortion in dairy herds.
A 2 to 1 matched case control study design was used to analyze herd level risk factors for Neospora caninum-associated abortion storms in 47 dairy herds. Data were obtained using a questionnaire regarding the state of affairs at the farms over the 2 years prior to the abortion storm. The questionnaire included 120 variables considered to be potential risk factors for either introduction of infection or recrudescence of chronic infection. The relationship between risk factors and case control pairs was analyzed by conditional logistic regression using a three-steps procedure. In addition, cross sectional serology was used to assess the possible role of concomitant infections. The main factors that were significant in the analysis and that were considered to have potential biological relevance were the presence of dogs, the presence of poultry, and the feeding of moldy maize-silage during summer. For both the presence of dogs and the presence of poultry on the farms, a linear relationship was found between the number of animals and the assessed risk for an abortion storm. These findings suggest a possible role of these species in the transmission of N. caninum. Further evidence for such a role of dogs was the significant association between the presence of dogs and the presence of seropositive cattle in the control herds. The feeding of moldy fodder is considered to be a factor which may induce recrudescence of a latent N. caninum-infection by mycotoxins causing immune suppression. We also found some evidence for a possible influence of management practices around calving and a high prevalence of retained afterbirths. No significant association was found for herd level prevalence of antibodies to bovine viral diarrhea virus, bovine herpesvirus 1, Leptospira hardjo or Salmonella dublin.
Abortion storms in 50 dairy herds in The Netherlands were reported in which there was a strong association with Neospora caninum-infection. The duration of the abortion storms ranged from 6 to 65 d (mean 41.5 d). The cumulative proportion of aborting cows ranged from 0.11 to 0.57 (mean 0.26) of the animals at risk. An apparent seasonal influence was noted as most abortion storms occurred during the summer and early autumn. The prevalence of antibodies to N. caninum in 50 herds which had had an abortion storm was compared with that of 100 control herds which had no history of an abortion storm. Seroprevalence was estimated by testing a 20% cross sectional herd sample using a tachyzoite lysate-based ELISA method. Seroprevalence in case herds (range 17 to 87%, mean 51.5%) was significantly higher than that in control herds (range 0 to 53%, mean 13.9%). For most herds the seroprevalence levels were equal across all age groups, which suggests that the infection had been perpetuated by vertical transmission. In these herds, the abortion storms appeared to be induced by factors causing recrudescence of a N. caninum-infection in chronically infected animals rather than being the result of a recent introduction. In 6 case herds the seroprevalence in the dairy cows was significantly higher than in the young stock, which may have been attributable to superimposed postnatal infection.
Since the identification of Neospora caninum in 1984 as a parasite separate from Toxoplasma gondii by Bjerkas et al., and its subsequent characterization and classification in 1988 by Dubey and co-workers, this parasite has attracted increasing attention, primarily as an important causative agent of abortion in cattle and neuromuscular disease in dogs, but also as a complementary model system to T. gondii for investigating the basic biology of intracellular parasitism. During November 11-14, 1999, the COST 820 Annual meeting (Vaccines against coccidioses) took place in Interlaken, Switzerland. Almost half of the papers presented at that meeting were on N. caninum and neosporosis, reflecting the increasing awareness of the importance of this parasite on part of the scientific community in Europe. On the occasion of the meeting, participants in this COST Action involved in Neospora research in Europe were asked to participate in this invited review in order to document the growing interest in N. caninum and the disease it causes. Thus, this paper is a unique collection of contributions provided by several European experts in the field. It is comprised of 10 reviews or original papers on different aspects of Neospora research including epidemiology, immunology, application and development of serological tools, and molecular characterisation of the parasite currently carried out throughout Europe. In addition, two distinguished invited speakers from overseas (Milton McAllister and John Ellis) provided valuable contributions. This invited review demonstrates that the COST 820 Action has brought together scientists from all over Europe and other parts of the world, and has laid the basis for many fruitful collaborations. The studies described here will contribute in assessing the relevance of neosporosis as a potential risk factor not only for animals, but also for human health.
Cattle immunised with a POLYGEN-adjuvanted killed Neospora caninum tachyzoite preparation were previously shown to produce interferon (IFN)-gamma at levels similar to those of tachyzoite-infected cattle. In view of the critical role of IFN-gamma in resistance of mice to N. caninum infection, these results prompted us to test the POLYGEN-adjuvanted preparation in pregnant cattle to determine whether it will be able to prevent foetal infection following an experimental tachyzoite challenge. Seven heifers were immunised at 35 and 63 days of gestation with the POLYGEN-adjuvanted preparation, while five heifers were inoculated with POLYGEN alone at the same days of gestation. Four weeks later, all heifers were challenged with a combined i.v./i.m. inoculation of tachyzoites. The same challenge was given to seven unimmunized heifers at the same stage of gestation. An additional unimmunized heifer was inoculated with uninfected monolayer cell culture material. All challenged heifers, immunized and unimmunized, had infected foetuses. Immunized heifers developed both parasite-specific humoral and cellular immune responses, characterised by increased IFAT titres, a predominant IgG1 response, elevated lymphoproliferative response and IFN-gamma production. Following tachyzoite challenge, they developed an anamnestic humoral response and produced similar amounts of IgG1 and IgG2 antibodies, but did not have an anamnestic cellular immune response. The lack of anamnestic cellular immune response and/or the large i.v/i.m tachyzoite inoculum may have contributed to the failure of the preparation.
In order to develop a vaccine against Neospora caninum in dogs, we constructed recombinant canine herpesvirus (CHV) expressing N. caninum surface protein, NcSRS2. Indirect immunofluorescence indicated that the antigenic structure of the recombinant NcSRS2 was similar to the authentic parasite protein. The dogs immunised with recombinant virus produced IgG antibody to N. caninum, and their sera recognised the parasite protein on Western blot. The dogs inoculated with recombinant virus showed no clinical symptoms and infectious CHV was not recovered from the dogs, suggesting that recombinant CHV expressing N. caninum proteins may lead to a vaccine against neosporosis in dogs.
Antibody titers to Neospora antigens ranged from 40 to 160 before vaccination, from 80 to 5,120 2 weeks after the first dose of vaccination, and 320 to 40,9602 weeks after the second (booster) vaccination. A peak antibody titer of 40,960 was also detected 28 days after the booster vaccination among animals vaccinated with Neospora vaccine formulated with Bay R1005 adjuvant. In heifers inoculated with experimental formulations of Neospora vaccines, transient development of injection site reactions resulted in 1 out of 15 animals. This injection site reaction was not detectable 14 days after the first observation and measurements were made. We have also demonstrated that vaccines derived from tissue-culture-grown Neospora tachyzoites are safe and would be expected to be efficacious.
The parasite, Neospora caninum is an important cause of abortion in cattle. It is transmitted vertically or horizontally and infection may result in abortion or the birth of a live, healthy but infected calf at full-term. Only a proportion of infected cattle abort and the pathogenesis of abortion is not understood. Groups of cattle were infected with 10(7) N. caninum tachyzoites intravenously at different times relative to gestation. Intravenous inoculation was chosen to reproduce the putative haematogenous spread of N. caninum following either recrudescence of endogenous infection or de novo infection. In all cattle, infection was accompanied by high gamma-interferon and lymphoproliferative responses, and a biased IgG2 response indicating that N. caninum infection is accompanied by a profound Th1 helper T cell-like response. Infection at 10 weeks gestation resulted in foetopathy and resorption of foetal tissues 3 weeks after infection in 5 out of 6 cows. Infection at 30 weeks gestation resulted in the birth of asymptomatic, congenitally-infected calves at full term in all 6 cows, whereas the 6 cows infected before artificial insemination gave birth to live, uninfected calves. These results suggest that the reason some cows abort is related to the time during gestation when they become infected or an existing infection recrudesces.
In order to develop a vaccine against Neospora caninum in dogs and cattle, we constructed a recombinant vaccinia virus expressing the N. caninum surface protein, NcSRS2 (Nc-p43). Monoclonal antibodies to NcSRS2 and anti-N. caninum tachyzoite mouse serum recognized the NcSRS2 expressed by the recombinant vaccinia virus. In addition, recombinant NcSRS2 was transported to the cell surface. Mice infected with the recombinant virus predominantly produced IgG1 antibody (Ab) to N. caninum, rather than producing IgG2a Ab. Moreover, splenocytes from mice infected with the recombinant virus proliferated in the presence of the N. caninum antigen. Mice immunized with the recombinant virus gave rise to humoral and cellular immune responses to N. caninum tachyzoites. This study showed that a recombinant vaccinia virus expressing NcSRS2 might be useful for the production of a live vaccine against N. caninum infection.
Neospora caninum infection is the major cause of bovine abortion. To develop a vaccine against N. caninum infection, recombinant vaccinia viruses carrying NcSRS2 and NcSAG1 genes (vv/Nc-p43 and vv/Nc-p36, respectively) were constructed and were tested in a mouse model. Vaccination of dams with vv/Nc-p43 appeared to confer effective protection against vertical transmission to offspring, though that with vv/Nc-p36 only provided partial protection. Moreover, the vv/Nc-p43 vaccination provoked cellular immune responses and antibody production against N. caninum. In conclusion, it is expected that vv/Nc-p43 can be used as an effective live vaccine to prevent vertical transmission of N. caninum in natural hosts.
The study describes the time course of the Neospora caninum-specific antibody response in experimentally infected foxes, in naturally N. caninum-seropositive vixens and their litters. An immunofluorescence test, a tachyzoite surface antigen based ELISA and an immunoblot assay were established for this purpose. The immunoblot patterns of naturally seropositive and experimentally infected foxes revealed a high degree of similarity and resembled those reported for other intermediate host species. Reactions against immunodominant antigens of Mr 56, 68 and >94 kDa were observed which could be linked with a period of 14 days-2 months post experimental infection with tachyzoites. Cubs born by naturally seropositive vixens were found to be persistently or transiently seropositive, in the latter case, specific antibodies were detected only up to 44 days after birth. These antibodies may thus be of maternal origin. Differences between the immunoblot patterns of persistently positive cubs, those of their mothers and of transiently positive cubs, in particular the differential response to antigens of Mr 56 and 68 kDa, prove that cubs with persistent antibodies had actively mounted an antibody response. This result provides the first evidence for the postnatal or vertical transmission of N. caninum among naturally seropositive foxes.
To evaluate efficacy of embryo transfer into seronegative recipients, using the procedure proposed by the International Embryo Transfer Society (IETS), for preventing vertical transmission of Neospora caninum in cattle.
Prospective clinical trial.
87 recipient cows and heifers and their embryo transfer calves from 22 donors originating from 9 dairy herds.
Neospora caninum serologic status of donors and recipients was determined before collection and transfer of embryos. Viable embryos were washed and treated with trypsin. Recipients in experimental groups A (n = 50) and B (29) were seronegative and received embryos from seropositive and seronegative donors, respectively. Recipients in group C (n = 8) were seropositive and received embryos from seronegative or seropositive donors. Antibody titers against N caninum were determined monthly during pregnancy in recipients and in calf blood samples collected at birth. Tissues collected from stillborn calves and aborted fetuses were analyzed histologically and by immunohistochemical (IHC) methods.
76 calves and 11 fetuses and stillborn calves were examined. All calves from groups A and B were seronegative (n = 70) or lacked evidence of infection by use of tissue analysis (9). In group C, 5 of 6 calves were seropositive at birth, and IHC results were positive for 1 of 2 calves. Vertical transmission rate was significantly lower in groups A and B (0%) than in group C (75%).
Embryo transfer into seronegative recipients, using the procedure proposed by IETS, is an effective way to prevent vertical transmission of N caninum. Results provide support for pretransfer testing of all embryo transfer recipients.
An experiment was carried out to determine whether bovine colostrum or placenta could be a source of infection of Neospora caninum for dogs. For this purpose, two dogs were fed bovine colostrum to which culture-derived N. caninum tachyzoites were added and two other dogs were fed placental cotyledonary tissue from N. caninum seropositive cows. One dog served as a negative control during the start of the experiment but this control dog was fed cotyledonary tissue later on. None of the dogs did produce serum antibodies to N. caninum. All three dogs that were fed cotyledonary tissue did shed N. caninum oocysts, but no oocyst shedding was seen in the two dogs that were fed colostrum with N. caninum tachyzoites. Oocyst excretion did not resume in two dogs after repeated feeding of N. caninum infected placenta. The identity of the oocysts was confirmed by a bioassay in gerbils. It is concluded that ingestion of bovine placenta by dogs is an effective mode of transmission of N. caninum from cattle to dogs.
Neospora caninum is a major cause of abortion in cattle worldwide. Cattle become infected with N. caninum by ingesting oocysts from the environment or transplacentally from dam to fetus. Experimentally, dogs can act as definitive hosts, but dogs excrete few oocysts after ingesting tissue cysts. A natural definitive host was unknown until now. In the present study, N. caninum was isolated from the feces of a dog. Gerbils (Meriones unguiculatus) fed feces from the dog developed antibodies to N. caninum in the Neospora caninum agglutination test, and tissue cysts were found in their brains. Neospora caninum was isolated in cell culture and in gamma-interferon gene knockout mice inoculated with brain homogenates of infected gerbils. The DNA obtained from fecal oocysts of the dog, from the brains of gerbils fed dog feces, and from organisms isolated in cell cultures inoculated with gerbil brains was confirmed as N. caninum. The identification of N. caninum oocyst by bioassay and polymerase chain reaction demonstrates that the dog is a natural definitive host for N. caninum.
A study was conducted with a 1998 retained-ownership population of Texas (USA) beef calves to determine the ranch-management practices associated with calf seroprevalence to Neospora caninum. Management practices of 76 Texas ranches that consigned 760 calves to a retained-ownership feedlot program were reviewed from a mailed questionnaire. Ninety-nine of 760 (13%; 95% CI, 9.4%, 17.7%) calves were positive to N. caninum and 59% of the ranches consigned at least one positive calf. In the logistic multiple-regression model which controlled for overdispersion, increased odds of calf-level seropositivity was associated with seasonal calving patterns, with stocking>1cow/calfunit/2.2ha, using a round-bale feeder, allowing wildlife access to the weaning supplement, and self-reared replacement heifers. However, decreased odds of seropositivity was associated with using a cattle-working dog and with using a self-contained cattle feeder. There was substantial overdispersion due to ranch.
In this study we were interested to determine whether infection of cattle prior to pregnancy would afford any protection to the foetus if the dams were challenged with Neospora caninum at mid-gestation. The experiment comprised four groups of cattle: group 1, uninfected controls; group 2, inoculated with N. caninum tachyzoites 6 weeks prior to mating and then challenged with N. caninum at mid-gestation; group 3, naive cattle challenged with N. caninum at mid-gestation and group 4 were infected with N. caninum prior to mating and left unchallenged throughout pregnancy. Positive cell-mediated and humoral immune responses to N. caninum were recorded in groups 2 and 4 prior to pregnancy and in groups 2, 3 and 4 following challenge at mid-gestation. However there was a marked down regulation of the cell-mediated immune response in all groups around mid-gestation. There was a significant increase in rectal temperature response in animals in group 3 compared to group 2 following challenge but no other clinical symptoms of disease were recorded and all cattle proceeded to calving. At calving, pre-colostral blood samples were negative for antibodies to N. caninum in all the calves born to dams in groups 1, 2 and 4. In contrast, all the calves born to dams in group 3 had high levels of specific antibody to N. caninum indicating that they had been exposed to the parasite in utero. At post-mortem N. caninum DNA was detected in CNS, thymus and placental cotyledon samples in calves from group 3. All tissue samples from calves in the other 3 groups were negative for N. caninum DNA with the exception of one calf from group 2 where specific DNA was detected in a sample of spinal cord. These results suggest that the immune response generated in the dams in group 2 prior to pregnancy had protected against vertical transmission of the parasite following challenge at mid-gestation.
In a previous paper we demonstrated that Hammondia heydorni-like oocysts isolated in 1996 from a naturally infected dog could not be distinguished from the isolate Neospora caninum NC-1. The isolate, designated as H. heydorni-Berlin-1996, was cyclically transmitted using dogs as the final hosts. The present study provides information on the antibody responses of the dogs used for the cyclical transmission of this isolate. The majority of dogs that had shed oocysts showed no sero-conversion with respect to N. caninum tachyzoite surface or immunodominant antigens, either in the indirect fluorescent antibody test or in two Western-blot-based tests. In addition to the examination of responses to immunodominant antigens, we also analysed the antibody reactions of dogs to a high-molecular-weight antigen (152 kDa) in the tachyzoite antigen preparation. The antibodies against this antigen appeared after the dogs had been fed infected intermediate host tissues and shed oocysts. The reaction was observed in dogs between day 35 and day 447 after feeding of intermediate host tissues. Therefore, our study provides initial information on a 152 kDa tachyzoite antigen, which might be a suitable candidate to identify dogs with a history of shedding N. caninum oocysts.
The prevalences of antibodies to the protozoan parasites Toxoplasma gondii and Neospora caninum were investigated by the direct agglutination test (DAT) and ELISA, respectively, in 221 red foxes (Vulpes vulpes) from different parts of Sweden. A total of 84 (38%) of the analysed sera had antibodies to T. gondii, but none of the foxes had antibodies to N. caninum. The results indicate that T. gondii infection is fairly common in Swedish red foxes and that the infection is present in most parts of the country. They also show that N. caninum is not widespread as a latent infection among red foxes in Sweden.
Neospora caninum is an intracellular apicomplexan parasite that infects a wide range of mammals and has been associated with abortion in cattle worldwide. Artemisinin is an effective antimalarial compound derived from a traditional Chinese herbal remedy, qinghao or Artemisia annua L. In the study reported, the cultured host cells (vero cells or mouse peritoneal macrophages) infected with N. caninum tachyzoites were incubated with alpha-MEM (minimal essential medium) 10%HS supplemented with various concentration or artemisinin (20, 10, 1, 0.1 and 0.01 microg/ml) to examine the efficacy of artemisinin against N. caninum tachyzoites intracellular multiplication. In long-term studies, at 20 or 10 microg/ml for 11 days, artemisinin reduced N. caninum and completely eliminated all microscopic foci of N. caninum. At 1 microg/ml for 14 days, artemisinin reduced N. caninum and completely achieved elimination of all microscopic foci of N. caninum. There was no apparent toxicity to host cells in long-term studies. In short-term studies, at > or = 0.1microg/ml, artemisinin reduced N. caninum tachyzoites intracellular multiplication, significantly (P < 0.05) and appeared to depend on the artemisinin concentrations. Pretreatment of host cells or N. caninum tachyzoites with artemisinin had no effect on N. caninum tachyzoites intracellular multiplication. These results demonstrate that artemisinin inhibited N. caninum tachyzoites intracellular multiplication.
Twelve dairy herds with evidence of post-natal infection with Neospora caninum were compared with 21 control herds with no evidence of post-natal infection. On the former farms, dogs consumed placenta or licked uterine discharge in 75 and 67% of the farms, respectively, while on control farms these activities occurred in 38 and 24% of the farms, respectively. On all control farms and all but three post-natally infected farms the dogs were fed colostrum or milk. Defecation of dogs on the feeding alley was observed in 92% of the post-natally infected farms and in 24% of the control farms. The same trend was observed for defecation of dogs in grass silage, in 75% of the post-natally infected farms and in 19% of the control farms; and in corn silage, in 50% of the post-natally infected farms and in 10% of the control farms. Consumption of placenta, material of aborted foetuses or uterine discharge in combination with defecation on the feeding alley, storage of grass or corn silage was observed in 19% of the control farms and in 75% of the post-natally infected farms. This study supports the hypothesis that farm dogs may become infected by foetal fluids or placental material of infected cattle, and may subsequently cause a post-natal infection of cattle in the herd by shedding oocysts.
Eight dairy herds with evidence of post-natal transmission of Neospora caninum were used to test the hypothesis of a point source exposure by a retrospective analysis of the housing and feeding of infected age-groups. The first N. caninum-associated abortion or birth of N. caninum-seropositive offspring from the post-natally infected age-group was considered as the first indication of the infection. In seven of the eight dairy herds, a point source exposure to N. caninum of the infected age-groups was found during a limited period of common housing and feeding. In all herds studied, the analysis indicated that the cattle had been infected shortly before the first abortions occurred. In all, except one herd, the post-natal infection was more directly related to housing than to feeding. Therefore, it appeared that the feed was contaminated in the feeding alley. In one herd, the total mixed ration was found to be the probable path of infection. In all farms studied, a new dog (young, adult dog or litter) had been introduced within a period of 1.5 years prior to the first indication of N. caninum infection in the cattle. As there was evidence in all herds of vertical transmission of neosporosis for years, it is hypothesized that the newly introduced dog was infected with N. caninum by materials from already infected cattle and subsequently transmitted the infection to other cattle by shedding of oocysts.
Parasite-specific antibody responses to Neospora spp. and Toxoplasma gondii, antigens were detected using the indirect fluorescent antibody test (IFAT) and immunoblot analysis in a korean equine population located on Jeju island, South Korea (126 degrees 12' E and 33 degrees 34' N). For comparison, a naturally infected Neospora hughesi horse and an experimentally inoculated T. gondii equid (pony) were used. In addition, all samples were tested for antibodies to Sarcocystis neurona by immunoblot analysis. A total of 191 serum samples from clinically normal horses were evaluated. Only 2% (4 out of 191) and 2.6% (5 out of 191) of the samples had showed reactivity at 1:100 using the IFAT for Neospora spp. and T. gondii, respectively. For T. gondii, two samples matched the antigen banding pattern of the positive control by immunoblot analysis. No sample was positive for N. hughesi by immunoblot analysis in this study. Overall, there was a 1% seroprevalence for T. gondii antibodies in the horses tested based on immunoblot analysis. The seroprevalence for S. neurona and N. hughesi antibodies was 0%. We concluded that these horses are either not routinely exposed to these parasites or antibody titers are not sufficiently elevated to be detectable. It is most likely the former explanation since Jeju island equine farms are isolated from the main land, and the horses were all less than 3 years of age. This naïve population of horses could be useful when evaluating S. neurona serodiagnostic tests or evaluating potential S. neurona vaccines since exposure risks to S. neurona and closely related parasites are negligible.