Low seroprevalence of Neospora caninum infection associated with the limousin breed in cow-calf herds in Andorra, Europe

Neospora caninum seroprevalence and risk factors affecting seroprevalence in beef cattle in Andorra were investigated. Antibodies to N. caninum were evaluated by enzyme-linked immunosorbent assay performed on a yearly basis in 1,758 animals older than 6 mo, belonging to 26 herds. Mean seroprevalence of antibodies to N. caninum for the herds was 7.4 +/- 1.2% (130/1,758). Logistic regression analyses were performed on data from each animal, considering N. caninum seropositivity as the dependent variable, and herd, grazing area, year of sampling, repeat-test animal (animals sampled twice or more), sex, breed, age (animals <4 yr old or > or =5 yr old), and country of birth as possible risk factors. Based on the odds ratio, the prevalence of infection was 2.1 times higher (P < 0.01) in animals from the Ordino grazing area, 1.64 times higher in animals older than 5 yr (P < 0.01), and 6.7 times (1/0.15) lower in Limousin-mixed Limousin cattle (P < 0.002). The results suggest that the particular grazing location could promote the horizontal transmission of this parasite and that certain breeds are less susceptible to N. caninum infection than others.     

Neospora caninum is associated with abortion in Algerian cattle

Neospora caninum is a major cause of abortion in cattle worldwide. However, little information is available for Algeria. Accordingly, 799 cattle from 87 farms in the north and northeast of Algeria were enrolled in a seroepidemiological survey. An indirect fluorescence antibody test (IFAT) revealed a seroprevalence of 19.6%. The animals were divided into 3 groups according to their breed: imported European cattle, local breeds, and crossed animals (European × local). Seroprevalences were 16.0%, 34.3%, and 18.6% in groups 1, 2, and 3, respectively. A case control study was performed to investigate the link between global seropositivity to N. caninum and abortion risk in those cattle farms. There was a significant (P < 0.01) association between the seroprevalence against N. caninum and the occurrence of abortion in those farms (odds ratio [OR] = 12.03). This was also observed at the individual level (OR = 2.79). The analysis of results according to the breed revealed a significant association between seroprevalence and abortion in groups 1 and 3, but not for group 2, despite the fact that the highest seroprevalence was observed in group 2. Cerebral tissues from 5 aborted fetuses were available for histology and polymerase chain reaction (PCR). One sample was found positive both by histology and by PCR, 2 samples were positive by PCR only, and 2 samples were negative in both tests.     

First isolation of Neospora caninum from an aborted bovine fetus in Spain

Neospora caninum was isolated from the brain of a 6-mo-old aborted bovine fetus from Galicia, Spain. The fetal brain homogenate was inoculated intraperitoneally into cortisonized mice. The peritoneal exudate from the infected mice, along with mouse sarcoma cells (Tg180), was inoculated into a second group of mice, and parasites were harvested from the peritoneal exudate. The parasites were adapted to in vitro growth in Vero monolayers. The tachyzoites from the peritoneal exudate reacted positively with anti-N. caninum antibodies and not with anti-Toxoplasma gondii antibodies on indirect fluorescent antibody test. The tachyzoites were lethal to interferon gamma gene knock out (KO) mice and could be identified immunohistochemically in the tissues. The identity of the parasite was also confirmed by polymerase chain reaction amplification of N. caninum-specific fragments. The sequences of the amplified gene 5 fragments (GenBank AY494944) were found to be identical to that of an Austrian isolate of N. caninum but not to that of NC-1. This is the first isolation of viable N. caninum from Spain.     

Transplacental transmission and abortion in cows administered Neospora caninum oocysts

Neospora caninum infection is a common cause of bovine abortion. One method by which cattle can acquire infection is through ingestion of oocysts; however, this has not yet been proved to cause transplacental infection or abortion. In this study, 19 cows, pregnant between 70 and 176 days, were administered 1500 to 115,000 oocysts through an esophageal tube. Seventeen of the cows became seropositive, indicating acquisition of infection, whereas 8 negative control cows remained seronegative (P < 0.001). Offspring were examined using serology, histology, immunohistochemistry, parasite isolation, and polymerase chain reaction (PCR). Six offspring were infected and 1 of them was aborted. The aborted fetus had typical lesions and positive immunohistochemistry and PCR for N. caninum. All 6 cows with infected offspring had continuously rising antibody titers, whereas 10 of 11 infected cows with uninfected offspring had falling titers after an early apex. The risk of transplacental transmission was increased by later exposure times during gestation and by the dose of oocysts (P < 0.01 for the 2 combined variables). The lowest dose of oocysts, when administered after the 160th day of gestation, caused transplacental infection in 1 of 2 animals. This study demonstrates that infection with N. caninum oocysts can cause transplacental transmission and abortion in cattle.     

Cow attributes,herd management,and reproductive history events associated with abortion in cow-calf herds from Western Canada

The primary objective of this study was to identify herd management and cow characteristics that are associated with abortion in cow-calf herds in Western Canada. Reproductive events were closely monitored in 29,713 cows in 203 herds from the beginning of the breeding season in 2001 through the calving season in 2002. Herd management and cow-level risk factors such as age, body condition score, and previous reproductive history were measured through a series of herd visits by project personnel and detailed individual animal records maintained by the herd owner. Pregnancy status was assessed in fall of 2001 by the herd veterinarian. Cows most likely to abort were replacement heifers, cows that were more than 10 years of age, cows with a body condition score of less than or equal to or 5 of 9 at pregnancy testing, or with twin pregnancies. Cows vaccinated for bovine viral diarrhea virus and infectious bovine rhinotracheitis and bred on community pastures were less likely to abort than cows from community pastures that were not vaccinated. Cows bred on community pastures that were not vaccinated were also more likely to abort than cows that were not on community pastures regardless of vaccination status. Adverse calving-associated events such as severe dystocia, problems such as uterine prolapse or retained placentas, abortion or calf death within 1 hour of birth were also associated with an increased risk of abortion the subsequent calving season after accounting for all other factors.     

First isolation of Neospora caninum from the feces of a naturally infected dog.

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.     

Seroprevalence of Neospora caninum infection in dairy and beef cattle in Spain

In recent years, neosporosis has been identified as a major cause of abortion in dairy and beef cattle. Although the disease has been described worldwide, there is a Jack of information concerning the prevalence of this infection in different cattle production systems. The aim of this study was to investigate the seroprevalence of Neospora caninum infection in a representative area of beef and dairy cattle production in Spain. A cross-sectional study was undertaken in which herds constituted the initial sampling unit and two strata (dairy and beef herds) were considered. Using a 95% level of confidence and setting 5% (beef) and 5.4% (dairy) error limits, 216 beef and 143 dairy herds were randomly selected and sampled. Nine animals (> 1 year old) were randomly sampled in each herd to detect the presence of the infection. A herd was considered infected when at least one animal was seropositive. In total, serum samples from 1121 dairy and 1712 beef animals were collected and tested for specific anti-N. caninum IgG using an ELISA. Specific antibodies were detected in 55.1% (119/216) beef and 83.2% (119/143) dairy herds. Individual prevalences obtained were 17.9% (306/1712) for beef and 35.9% (402/1121) for dairy animals. Presence of N. caninum infection was higher in dairy than in beef herds and the association between infection and the cattle production system (dairy or beef) was statistically significant [(chi2)Y= 29.21, P < 0.001, OR = 4.04 (2.35-6.99)]. Herd size of dairy cattle did not appear to be associated with N. caninum infection. On the contrary, infection was associated with herd size in beef cattle (chi2 = 12.79, P < 0.01). Finally, no association was found between replacement or pasture management and infection in beef herds.     

GRA12, a novel dense granule protein from Neospora caninum

Neospora caninum, an obligate intracellular parasite of the phylum Apicomplexa, is a major cause of abortion in cattle. After invasion, tachyzoites can reside in the parasitophorous vacuole (PV) and ingest nutrition through the intravacuolar network (IVN). Secreted dense granule proteins of N. caninum (NcGRAs) may play important roles in maintaining the structures of the PV and IVN. In this study, we predicted a NcGRA12 gene; aligned it with Toxoplasma gondii GRA12 for homology analysis; and analyzed the ORF, signal peptide and transmembrane domain. Then, we cloned the NcGRA12 gene, expressed the NcGRA12 protein, prepared polyclonal antibodies, and carried out colocalization analysis of NcGRA12 with NcGRA6 in extracellular tachyzoites and intracellular PVs using an immunofluorescence assay (IFA). Finally, we determined the solubility of the NcGRA12 protein. The results showed that NcGRA12 shared 59.13% nucleotide homology and 44.9% amino acid homology with TgGRA12. There was no predicted signal peptide or transmembrane domain. IFA data of extracellular tachyzoites showed that the NcGRA12 protein was secreted by the apical organ and located at the posterior end of tachyzoites, which was consistent with TgGRA12. IFA data of intracellular PVs identified NcGRA12 in the IVN membranes. Moreover, NcGRA12 could colocalize with NcGRA6 in intracellular PVs but not extracellular tachyzoites. Solubility analysis showed that NcGRA12 existed in soluble and membrane-related forms in the PV. Overall, we provide the first report of the novel NcGRA12 protein and verify that it is associated with the IVN membranes of PVs in N. caninum. These data lay a foundation for further research into the function of NcGRA12.     

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.     

A stochastic model for disease transmission in a managed herd, motivated by Neospora caninum amongst dairy cattle

A stochastic model for the spread of Neospora caninum infection within a herd of dairy cattle is studied, in particular the long-term (equilibrium) behaviour of the model. The model incorporates the interesting feature that total herd size is constrained to lie within a fairly small interval, but not held exactly constant. Approximations for the joint distribution of numbers of susceptible and infected individuals present in equilibrium are derived based upon a diffusion approximation to the infection process. The effect of both 'typical herd size' and 'the amount of permitted variation in herd size' upon disease prevalence in equilibrium are considered using both the exact equilibrium distribution of the process and our approximations.     

Increased risk of abortion following Neospora caninum abortion outbreaks: a retrospective and prospective cohort study in four dairy herds

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.     

Fatal congenital Neospora caninum infection in a lamb

Neospora caninum tissue cysts were found in the brain and spinal cord of a 1-wk-old lamb that was unable to stand after birth. The lamb was originally diagnosed as having toxoplasmosis, but ultrastructural and immunohistochemical techniques used in the present study permitted a definitive diagnosis of Neospora caninum tissue cysts in the brain and spinal cord of this lamb. This is the first report of N. caninum in sheep.     

Seroprevalence of antibodies to Neospora caninum in dogs from Spain

The prevalence of Neospora caninum antibodies was determined in sera of 139 dogs from Catalonia (northeastern Spain) using the indirect immunofluorescence antibody test (IFAT). Antibodies in the IFAT were found in 17 of 139 dogs (12.2%) with titers ranging from 1:50 to 1: 1,600. Seroprevalence was higher in dogs over 1 yr old compared with dogs younger than 1 yr (P < 0.05). No statistical difference was observed when sex, breed, purpose, or modus vivendi was compared with seropositivity. Most dogs had low antibody titers, which indicated subclinical infection in the area studied. No neosporosis-related disease was reported from any dog, although a German shepherd with an antibody titer of 1:800 showed pododermatitis. All sera were also screened using a commercial direct agglutination test (DAT). The DAT showed a similar specificity but a lower sensitivity when compared with IFAT as a reference technique.     

Seroprevalence of Neospora caninum and Brucella abortus in dairy cattle herds with high abortion rates

The protozoan Neospora caninum and the bacterium Brucella abortus are well-recognized causes of abortion in dairy cattle. Serum samples (n = 240) from aborting (n = 141) and at-risk (n = 99) animals from 5 herds with high abortion rates in Punjab Province, Pakistan, were tested for antibodies to N. caninum using monoclonal antibody-based ELISA and for antibodies to B. abortus using the serum agglutination test. Antibodies to N. caninum and B. abortus were detected in 105 (43.8%) and 135 (56.3%) cattle, respectively. Prevalences of antibodies to N. caninum and B. abortus were higher in aborting cows (46.8% and 76.6%, P < 0.05) than in animals at risk (39.4% and 27.3%, P > 0.05). Sixty-six animals (27.5%) were seropositive to both N. caninum and B. abortus , and results showed no significant difference (P > 0.05) with respect to geographical district, breed, and age. This is the first report of N. caninum infection among dairy cattle herds in Pakistan.     

Neospora caninum: comparative gene expression profiling of Neospora caninum wild type and a temperature sensitive clone

To understand the genetic basis of virulence, gene expression profiles of a temperature-sensitive clone (NCts-8, relatively avirulent) and its wild type (NC-1) of Neospora caninum were characterized and compared using a high-density microarray with approximately 63,000 distinct oligonucleotides. This microarray consists of 5692 unique N. caninum sequences, including 1980 Tentative Consensus sequences and 3712 singleton ESTs from the TIGR N. caninum Gene Index (NCGI, release 5.0). Each sequence was represented by 11 distinct 60mer oligonucleotides synthesized in situ on the microarray. The results showed that 111 genes were significantly repressed and no up-regulated genes were identified in the NCts-8 clone. The level of 10 randomly selected genes from the repressed genes was confirmed using real-time RT-PCR. Of the 111 repressed genes, 58 were hypothetical protein products and 53 were annotated genes. Over 70% of the repressed genes identified in this study are clustered on five chromosomes (I, VII, VIII, X and XII). These results suggest that the down-regulated genes may be in part responsible for the reduced pathogenesis of NCts-8; further characterization of the regulated genes may aid in understanding of molecular basis of virulence and development of countermeasures against neosporosis.     

First report of Neospora caninum infection in adult alpacas (Vicugna pacos) and llamas (Lama glama)

Neospora caninum is a cyst-forming coccidian that mainly affects bovines, although Neospora infection has also been described in other domestic and wild ruminant species. Serum samples from 78 alpacas (Vicugna pacos) and 73 llamas (Lama glama) at a unique dilution of 1:50 tested by indirect fluorescent antibody test (IFAT) were further analyzed serologically by IFAT and Western blot in both ruminant species to avoid cross-reactions with closely related coccidian parasites and to confirm the existence of N. caninum-specific antibodies. IFAT titers ranging between 1:50 and 1:800 were found. When using Western blot, N. caninum tachyzoite-specific immunodominant antigens with apparent molecular weights of 17-18, 34-35, 37, and 60-62 kDa were also recognized, although some sera with 1:50 IFAT titers proved not to have N. caninum-specific antibodies. As expected, higher IFAT titers were associated with higher anti-N. caninum reactivity in Western blot. This report documents for the first time the presence of N. caninum infection in adult alpacas and llamas from Peru.     

First demonstration of protective immunity against foetopathy in cattle with latent Neospora caninum infection

The parasite Neospora caninum is an important cause of abortion in cattle world-wide. Chronically infected dams transmit the parasite transplacentally and infected foetuses may be aborted or born chronically infected but clinically normal. Chronically infected cows repeatedly transmit the parasite to foetuses in several pregnancies and some may abort more than once suggesting that the immune response in these cattle is compromised during pregnancy. To investigate the nature of the immune response in chronically infected cattle, five naturally, chronically infected cows were challenged with N. caninum tachyzoites at 10 weeks of gestation. No foetopathy occurred and all five delivered live calves at full-term. In four naive pregnant cows challenged at the same time, all four foetuses died within 3-5 weeks of challenge. Of the five live calves born to the chronically infected challenged cows, three were transplacentally infected with N. caninum. The kinetics of the maternal anti-N. caninum antibody responses during gestation suggested that these transplacental infections were not the result of the superimposed challenge, but the result of the recrudescence of the maternal chronic infection-which occurred concurrently in non-challenged, chronically infected pregnant controls. These data provide the first experimental evidence that protective immunity occurs in neosporosis. They also suggest that whilst immunity to a pre-existing infection will protect against an exogenous challenge, this protective immunity will not prevent transplacental infection. This implies that a subtle form of concomitant immunity exists in chronically infected cattle and has important implications for vaccine development.     

Neospora caninum and Toxoplasma gondii antibodies in dogs from Durango City, Mexico

Toxoplasma gondii and Neospora caninum are structurally similar parasites, with many hosts in common. The prevalence of antibodies to T. gondii and N. caninum was determined in sera from dogs from Durango City, Mexico. Using a modified agglutination test, antibodies to T. gondii were found in 52 (51.5%) of the 101 dogs with titers of 1:25 in 27, 1:50 in 11, 1:100 in 5, 1:200 in 4, 1:400 in 2, 1:800 in 2, and 1:3,200 or higher in 1. Antibodies to N. caninum were determined by the indirect immunofluorescent antibody test (IFAT) and the Neospora sp. agglutination test (NAT). Two of the 101 dogs had N. caninum antibodies; these dogs did not have T. gondii antibodies, supporting the specificity of the tests used. The N. caninum antibody titers of the 2 dogs were: 1:400 by IFAT and 1:200 by NAT in 1, and 1:25 by NAT and IFAT in the other. Results indicate that these 2 structurally similar protozoans are antigenically different.