Neospora caninum (Apicomplexa) in an aborted equine fetus

Tachyzoites of Neospora caninum were found in sections of lung of an equine fetus aborted 2 mo before term. Individual tachyzoites were approximately 3-5 x 2-3 microns, divided by endodyogeny, and stained positively with anti-N. caninum serum but not with anti-Toxoplasma gondii serum. Toxoplasma gondii antibody was not found in the mare's serum. This is the first report of N. caninum in a horse and indicates that N. caninum can be transmitted transplacentally in equids.     

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.     

Development of a rickettsia isolated from an aborted bovine fetus.

An obligate intracellular rickettsial organism isolated from an aborted bovine fetus was studied in bovine turbinate and mouse macrophage cell cultures with light and electron microscopy. Development of the organism was similar in both cell types. The organism replicated within cytoplasmic vacuoles in a developmental cycle that resembled that of both the ehrlichiae and chlamydiae. The inoculum contained only electron-dense forms, which infected cells within 2 h postinoculation by adhering to cell membranes at thickened areas that appeared to be coated pits and then being endocytosed. A striking feature occurred next as the organisms became surrounded by host cell mitochondria and, by light microscopy, appeared to have halos. During this intimate association with mitochondria, the electron-dense organisms changed into large reticulated forms that began to divide by binary fission. These large forms were often in direct contact with mitochondrial membranes. The organisms continued to divide by binary fission, and host cells contained large cytoplasmic inclusions of reticulated organisms. The reticulated organisms gradually changed into electron-dense forms that were released from degenerated host cells.     

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.     

Influence of the stage of pregnancy on Neospora caninum distribution, parasite loads and lesions in aborted bovine foetuses

In the present work we have studied in Neospra caninum aborted bovine foetuses the influence of foetal age (first, second and third gestational periods) on parasite distribution by nested PCR, parasite loads by real-time PCR and N. caninum associated lesions. For this purpose, a total of 220 aborted foetuses were analysed and detection of N. caninum infection was accomplished by nested-PCR in brain, heart and liver, detecting the presence of the parasite in 72 (32.7%) bovine foetuses. When the different age classes were compared, parasite DNA-detectability in heart and liver was reduced over time of gestation (P < 0.05, Fisher F-test). N. caninum distribution, parasite loads and lesions were studied on 34 out of 72 N. caninum-infected foetuses selected according to the stage of pregnancy and organs recovered. A higher number of positive-PCR tissue samples were observed in the foetuses corresponding to the first and second pregnancy periods. In the last trimester, the parasite could only be detected in the brain and, sporadically, in the diaphragm, heart and lymph nodes. The parasite loads decreased during pregnancy and the foetuses from the first period had higher parasite burdens in brain, heart, kidney and lung (P < 0.05, Kruskal-Wallis H-test) than in those corresponding to the other two trimesters of pregnancy. In addition, the observed lesions were more severe in foetuses from the first and second pregnancy periods than those from the third period (P > 0.05, Kruskal-Wallis H-test). Our results confirm the influence of N. caninum foetal age on pathogenesis in natural N. caninum infections.     

Isolation and genetic characterization of Neospora caninum from asymptomatic calves in Spain

Neospora caninum is a cyst-forming parasite that causes abortion in cattle. Despite this parasite's ubiquitous distribution and wide host range, the number of N. caninum isolates obtained to date is limited. In vitro isolation of the parasite is arduous and often unsuccessful. In addition, most isolates have been obtained from clinically affected hosts and therefore could be biased towards more virulent isolates. In this report, an improved isolation approach from transplacentally infected newborn calves was undertaken and 9 new isolates were obtained. Moreover, a microsatellite technique was applied to investigate the genetic diversity of these isolates. Most isolates showed specific genetic profiles. However, the Nc-Spain10 isolate was identical to the previously described Nc-Spain1H isolate and Nc-Spain3H was identical to Nc-Spain4H. These isolates were likely to have identical genotypes because they were isolated from distinct calves of the same herd. Future pathogenic characterization of these isolates will contribute to the investigation of the relationship between isolate virulence and the outcome of infection, as well as other epidemiological features, such as transmission.     

Canine and bovine Neospora caninum control sera examined for cross-reactivity using Neospora caninum and Neospora hughesi indirect fluorescent antibody tests

Neospora caninum is a well known protozoan parasite of domestic and wild animals. Neospora hughesi is a closely related protozoan with an unknown life cycle, host range, and infection prevalence. Many serologic surveys of N. caninum have been performed without consideration of potential cross-reactions with N. hughesi, which could confound results. The aim of this study was to investigate whether postexposure sera from animals experimentally infected with N. caninum exhibit significant reactivity differences when tested using N. caninum and N. hughesi Immunofluorescent Antibody Tests (IFAT). Pre- and postinfection serum samples from 10 dogs, 20 calves, and 17 cows were tested by dual IFATs. All pre-exposure samples for N. caninum tested seronegative for both organisms. All postexposure samples that were seropositive for N. caninum were also positive for N. hughesi, although N. hughesi antibody titers were usually 1 dilution lower (P < 0.02). Serologic surveys for N. caninum may be confounded by cross-reacting titers with N. hughesi, but true positive N. caninum antibody titers are greater than, or equal to, cross-reacting N. hughesi antibody titers.     

Evaluation of Neospora caninum serodiagnostic antigens for bovine neosporosis

Abortion and reproductive failure caused by Neospora caninum infection has a dramatic negative economic impact on the cattle industry. To date, no definitive serodiagnostic tool for assessing N. caninum abortion has been reported. In this study, we evaluated the diagnostic performance of numerous N. caninum antigens in relation to abortion in cattle. Five recombinant proteins with potential as diagnostic antigens (NcGRA6, NcGRA7, NcGRA14, NcCyP, and NcSAG1) were compared by indirect enzyme-linked immunosorbent assay (iELISA) using sera from mice and cattle experimentally infected with N. caninum. The best-performing three antigens (NcSAG1, NcGRA7, and NcGRA6) were evaluated by IgG-iELISAs to assess their utility in diagnosing Neospora abortion using sera from confirmed N. caninum-aborted dams based on immunohistochemical assays (IHC). Additionally, all samples were tested using a commercial N. caninum antibody competitive ELISA (cELISA). The iELISAs against both NcSAG1 and NcGRA7 could efficiently distinguish IHC positive and negative samples compared with iELISAs against NcGRA6 and the cELISA. Furthermore, antibody levels against NcSAG1 and NcGRA7 were significantly higher in aborting cows comparing with infected but non-aborted dams in a herd experiencing a Neospora abortion outbreak. Tracking the dynamics of antibody levels during pregnancy revealed a marked increase in NcSAG1- and NcGRA7-specific antibodies at the last trimester of pregnancy. In contrast, no marked differences in antibody levels against either antigen were noted in neurologically symptomatic calves compared with non-symptomatic infected calves. Our data suggests NcSAG1 and NcGRA7 as indicators for Neospora abortion.     

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.     

Neospora caninum in wildlife

Neosporosis, which is caused by the coccidian parasite Neospora caninum, is recognized as a major disease of domestic animals that causes high abortion rates in cattle and fatal neurological disease in dogs. A life cycle of N. caninum in wild animals (i.e. sylvatic) has long been suspected because neosporosis has been detected in several wildlife species. Recently, the transmission of N. caninum has been confirmed in coyotes and white-tailed deer. The newly confirmed wild hosts and other wild animals are probably involved in the sylvatic cycle of the parasite. Control measures for neosporosis could now become more complicated, given the participation of wildlife in the life cycle of N. caninum.     

First report of Neospora caninum abortion in a beef cow-calf herd from Andorra, Europe

Neospora caninum-associated abortion was diagnosed in a 7-mo gestational age beef cow fetus from Andorra. The fetus had a multifocal necrotizing encephalitis and nonpurulent multifocal myocarditis. The diagnosis was confirmed by demonstration of N. caninum DNA by polymerase chain reaction and tachyzoites by specific staining with N. caninum polyclonal antibodies in the fetal brain. The dam of the aborted fetus had serum N. caninum antibodies at the time of abortion but not 2 mo before abortion took place. This is the first report of N. caninum abortion in Andorra and the first confirmed N. caninum abortion in an acutely infected cow.     

Dogs shed Neospora caninum oocysts after ingestion of naturally infected bovine placenta but not after ingestion of colostrum spiked with Neospora caninum tachyzoites

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 infections in bovine foetuses and dairy cows with abortions in Argentina

Antibodies to Neospora caninum were measured in bovine foetuses, dairy cows and beef cows in Argentina using the IFAT, the N. caninum agglutination test, and the recombinant NCDG1 and NCDG2 ELISA. Serum antibodies (IFAT titre 1:80) were found in 20 of 82 (24.4%) dairy cow foetuses and one of 22 (4.5%) beef cow foetuses. Microscopic lesions suggestive of neosporosis were seen in brains of seven of eight foetuses with IFAT titres of 1:80. Antibodies (IFAT) were found in 122 of 189 (64.5%) dairy cows that aborted. Serum antibody titres (IFAT) of 189 dairy cows that aborted were: < 1:25 (67 cows), 1:25 (four cows), 1:50 (16 cows), 1:200 (seven cows), 1:> or = 800 (95 cows). Of the 87 sera with IFAT titres of < or = 1:50, 57 had no antibodies in 1:40 dilution and 30 had titres of 1:40 in the N. caninum agglutination test. Thus, sera from at least 56 dairy cows which had aborted were seronegative both in the N. caninum agglutination test and the IFAT. The distribution of positive and negative sera was similar when measured by ELISA, except that, depending on cut-off titre, the ELISA indicated a greater number of seropositive cows that were negative by the IFAT and N. caninum agglutination test. These results suggest that transplacental transmission of N. caninum in dairy cows in Argentina is frequent.     

First isolation of Prototheca species in Spain

One-hundred forty samples of waste water were studied in search of algae of the genus Prototheca, that are potentially pathogenic to man. The samples were taken from collector outlets opening onto the Guadalquivir River.Nineteen cultures of Prototheca were isolated and identified as P. wickerhamii 9, P. zopffi 8 and P. stagnore 2.This is the first report of these algae in Spain and it is to be anticipated that these opportunist human pathogens will eventually be found to cause disease in the general population.     

Neospora caninum antigens defined by antigen-dependent bovine CD4+ T cells

Neosporosis is an important cause of pregnancy loss in cattle worldwide. The objective of the present study was to identify Neospora caninum antigens as vaccine candidates using antigen-specific, short-term CD4+ T cells established from N. caninum-immunized and -challenged cows. Whole N. caninum tachyzoite lysate was separated into 6 fractions by DEAE anion-exchange chromatography using high-pressure liquid chromatography (HPLC). The CD4+ T-cell proliferation assay results indicated that antigenic activity was associated with proteins from HPLC fractions 4-6, with fraction 5 exhibiting the highest antigenic activity. Also, SDS-PAGE analysis revealed a 16-kDa protein in fractions 4-6 that was recognized by anti-N. caninum antibodies. This 16-kDa protein was absent in other fractions, and it may be a target of a T-cell response in cattle. Further identification of immunogenic proteins of N. caninum may facilitate development of subunit vaccines against neosporosis.     

Transplacental Neospora caninum infection in cats

Transplacental transmission of Neospora caninum was studied in 2 pregnant cats (queens). Queen 1 was inoculated subcutaneously with 2 x 10(6) cell culture-derived N. caninum tachyzoites on day 47 of gestation. She gave birth to a full-term kitten on the 17th day after inoculation. The kitten died the second day after birth due to generalized N. caninum infection. The mother cat was killed on the third day after parturition and was found to have a macerated kitten in the uterus. Severe placentitis, metritis, hepatitis, and nephritis due to N. caninum were seen in tissues from the queen. Queen 2 was fed N. caninum tissue cysts and mated 111 days later. She gave birth to 3 healthy full-term kittens. The kittens were necropsied at 2, 22, and 30 days of age. Neospora caninum was recovered from the organs and was seen in histologic sections in 1 of the 3 kittens. Results indicate that N. caninum can be transplacentally transmitted in cats during acute and chronic stages of infection. Neospora caninum-specific IgG antibodies were demonstrated in the sera of inoculated cats and nursing kittens.     

Fatal Neospora caninum infection in kittens

Three 3-day-old kittens were inoculated subcutaneously and orally with Neospora caninum tachyzoites. A littermate and the queen were not inoculated with N. caninum and served as controls. Kitten 1 died between 14 and 16 days postinoculation (DPI) and was eaten by the mother. Kitten 2 died 17 DPI and kitten 3 was euthanized 29 DPI in a moribund condition. The control littermate and the dam remained healthy. Granulomatous skeletal myositis and nonsuppurative encephalomyelitis were the main lesions and were associated with numerous N. caninum tachyzoites in kittens 2 and 3. Cysts were found in kitten 3. Oocysts were not found in any cats. Neither lesions nor parasites were found in control cats.     

Neospora caninum: quantification of DNA in the blood of naturally infected aborted and pregnant cows using real-time PCR

This study quantified Neospora caninum DNA in the blood and brain of pregnant and aborted heifers by monitoring PCR product formation in real-time using SYBR Green I, a double-stranded DNA-binding dye. Primers were designed to amplify a 188 bp product specific to N. caninum from the Nc-5 gene fragment of N. caninum. Similarly, a 71 bp product was amplified from the 28S rRNA gene of bovine genomic DNA that served as a control. Agarose gel electrophoresis and analysis of the melting curve for PCR products showed that both primer pairs were specific to their targets. Standard curves were generated for both bovine and N. caninum genomic DNA, and were used to compute the relative concentration of parasite to bovine DNA in the test samples. The concentration of N. caninum DNA in 1 ng of bovine genomic DNA obtained from blood ranged between 0.097 ng at the 1st week of the observation and 0 ng at the 15th week in aborted cows. In pregnant cows, the values ranged between 0.080 ng at the 1st week and 0.155 ng at the 15th week of observation. There was a sustained decrease of DNA concentration in the aborted group after abortion and an increase in DNA concentration in the pregnant group. Comparison of parasite DNA in blood and brain of infected heifers showed a higher DNA concentration in brain than in blood. This study shows that N. caninum DNA can be quantified to obtain the relative concentration of parasite DNA to host genomic DNA in blood. This technique allows testing of a large number of samples at one time and can be done without the need for slaughter of tested animals.     

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 emerges from the shadow of Toxoplasma gondii

It is sometimes easy to make the mistake of assuming that everything that holds true for Toxoplasma gondii is also true for its relative Neospora caninum. However, a recurring theme in the recent review by Hemphill et al. is not the similarities but the striking differences between the two parasites.