Experimental infection of sheep with Neospora caninum oocysts

The purpose of the present study was to investigate the potential of Neospora caninum oocysts to infect sheep and determine whether N. caninum DNA could be detected by polymerase chain reaction (PCR) assay in blood and brain of sheep after oocyst inoculation. Six ewes were inoculated per os with 10(4) N. caninum oocysts, whereas 2 ewes served as uninoculated controls. All sheep were bled weekly for 7 wk after inoculation. Blood was analyzed for the presence of N. caninum DNA by 2 different PCR assays, as well as for the presence of antibodies to recombinant and native N. caninum antigens. Neospora caninum DNA was detected in 2 sheep as early as 7 days after oocyst inoculation (DAOI). All 6 sheep were PCR positive by 32 days and remained positive until the end of the study at 49 DAOI. Aside from 1 ewe, all sheep inoculated with N. caninum oocysts contained detectable N. caninum DNA in the brain tissue collected at 49 DAOI. Unlike with PCR, no lesion or parasite was detected by immunohistochemistry. Antibodies were detected by enzyme-linked immunosorbent assay, Neospora agglutination test, or immunoblotting to either native or recombinant N. caninum antigens in sheep inoculated with oocysts.     

Improved production of Neospora caninum oocysts,cyclical oral transmission between dogs and cattle,and in vitro isolation from oocysts

Scarce information is available about Neospora caninum oocysts and sporozoites, in part because only small numbers of oocysts have typically been produced by experimentally infected dogs. We hypothesized that I reason for low experimental production of oocysts is that dogs have been fed tissues from experimentally infected mice instead of tissues from cattle (which are natural intermediate hosts of N. caninum). In this study, 9 dogs were fed tissues from N. caninum-infected calves, and oocyst production was compared with 6 dogs that were fed infected mouse carcasses. The number of oocysts produced by dogs that ingested infected calf tissues (mean = 160,700) was significantly greater (P = 0.03) than the number of oocysts shed by dogs that ingested infected mice (mean = 5,400). The second goal of our experiment was to demonstrate cyclical oral transmission of N. caninum between dogs and cattle. As few as 300 oocysts were used to successfully infect calves, and tissues from these calves induced patent infections in 2 of 3 dogs; oocysts from I of these dogs were administered to another calf, and tissues from this calf subsequently induced a third dog to shed oocysts. Oocysts were confirmed to be N. caninum using a species-specific polymerase chain reaction technique. In addition, sporulated oocysts were used to recover N. caninum in vitro after digestion in an acid-pepsin solution and inoculation of cell monolayers.     

Specific detection of Neospora caninum oocysts in fecal samples from experimentally-infected dogs using the polymerase chain reaction

Neospora caninum oocysts, passed in the feces of a definitive host (dog), were isolated, and genomic DNA was extracted. A polymerase cahin reaction (PCR) targeting the N. caninum-specific Nc 5 genomic sequence was performed using the isolated DNA. A synthesized competitor molecule containing part of the Nc 5 sequence was included in the assay as a check against false-negative PCR results and to quantify N. caninum oocyst DNA in fecal samples. A standard curve of the ratio of fluorescence intensity of PCR-amplified competitor to that of oocyst DNA was constructed to compare oocyst equivalents from fecal samples containing unknown numbers of N. caninum oocysts and to assess the sensitivity of the assay. The specificity of the assay was determined using the Nc 5-specific primers in PCR assays against other parasites likely to be found in canine feces. Genomic DNA sequences from the canine coccidians Hammondia heydorni, Cryptosporidium parvum, Sarcocystis cruzi, S. tenella, and Isospora ohioensis and the canine helminth parasites Strongyloides stercoralis, Toxocara canis, Dipylidium caninum, and Ancylostoma caninum were not amplified. In addition, genomic DNA sequences from oocysts of coccidian parasites that might contaminate dog feces, such as Hammondia hammondi, Toxoplasma gondii, or Eimeria tenella, were not amplified in the PCR assay. The assay should be useful in epidemiological surveys of both domestic and wild canine hosts and in investigations of oocyst biology in experimental infections.     

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.     

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.     

Protective efficacy of vaccination with Neospora caninum multiple recombinant antigens against experimental Neospora caninum infection

Protective efficacy of vaccination with Neospora caninum multiple recombinant antigens against N. caninum infection was evaluated in vitro and in vivo. Two major immunodominant surface antigens (NcSAG1 and NcSRS2) and two dense granule proteins (NcDG1 and NcDG2) of N. caninum tachyzoites were expressed in E. coli, respectively. An in vitro neutralization assay using polyclonal antisera raised against each recombinant antigen showed inhibitory effects on the invasion of N. caninum tachyzoites into host cells. Separate groups of gerbils were immunized with the purified recombinant proteins singly or in combinations and animals were then challenged with N. caninum. Following these experimental challenges, the protective efficacy of each vaccination was determined by assessing animal survival rate. All experimental groups showed protective effects of different degrees against experimental infection. The highest protection efficacy was observed for combined vaccination with NcSRS2 and NcDG1. Our results indicate that combined vaccination with the N. caninum recombinant antigens, NcSRS2 and NcDG1, induces the highest protective effect against N. caninum infection in vitro and in vivo.     

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.     

Dirofilaria immitis: Parasite-specific humoral and cellular immune responses in experimentally infected dogs

Humoral and cellular immune responses to adult antigens of Dirofilaria immitis were evaluated in experimentally infected dogs during the chronic phase of infection. All infected dogs had significantly elevated IgG (enzyme-linked immunosorbent assay) and IgE (passive cutaneous anaphylaxis) titers against D. immitis adult antigens. However, there was little difference between infected dogs and uninfected controls in cellular-immune responses to D. immitis adult antigen or phytohemagglutinin as assessed by the lymphocyte transformation assay. Although neither cellular nor humoral responses correlated with worm burdens, cellular responses among infected dogs correlated inversely with IgG titers to D. immitis adult antigen. These results are consistent with observations in other nematode and trematode systems which suggest that in chronic tissue helminth infections there is suppression of cellular immune responses to parasite antigens while humoral responses to the same antigens remain relatively preserved.     

Serological diagnosis of Neospora caninum infection

Since the first isolation of the apicomplexan parasite Neospora caninum, a range of serological assays have been developed for use in dogs, cattle and a variety of other potential host species. The tests include the indirect fluorescent antibody test, the direct agglutination test and different enzyme-linked immunosorbent assays. This article reviews the principles and properties of the available tests which are discussed in relation to different applications.     

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.     

Natural killer cells act as early responders in an experimental infection with Neospora caninum in calves

The intracellular protozoan parasite Neospora caninum is a cause of abortion and congenital disease in cattle worldwide. We have previously shown that natural killer (NK) cells produce IFN-gamma in response to N. caninum tachyzoites in vitro. This study aimed to investigate the role of NK cells and other cellular immune responses in an experimental N. caninum infection model in calves. Phenotyping of peripheral blood lymphocytes showed a drop in the percentage of NK cells at days 4-6 after i.v. inoculation, followed by an increase in the percentage of both NK cells and CD8+ T cells which peaked at days 11-15. A whole blood flow cytometric assay showed that CD4+ T cells were the major IFN-gamma producing cells, but in the early stages of the infection both NK cells and CD8+ T cells contributed to IFN-gamma production. We also compared the ability of two different N. caninum antigen preparations--sonicated soluble antigens and intact heat-inactivated parasites--to induce proliferation and IFN-gamma production in various cell types. Heat-inactivated tachyzoites induced a 3.7 times greater increase in the number of IFN-gamma producing NK cells compared with sonicated soluble antigens. This indicated the presence of some NK cell-stimulating antigens in the intact tachyzoite that were absent from the sonicated soluble antigens. The heat-inactivated whole tachyzoites also inhibited gammadelta T cell proliferation while the soluble antigens from N. caninum did not. We believe this is the first time NK cells have been demonstrated to be early responders in N. caninum infection in calves.     

In vitro development of Neospora caninum (Protozoa: Apicomplexa) from dogs

The development of Neospora caninum isolated from naturally infected dogs was examined in mammalian cell cultures. Tachyzoites developed by endodyogeny when inoculated onto bovine monocyte or bovine cardiopulmonary artery endothelial cell cultures. Tachyzoites were 5.0 by 2.0 microns and had a posteriorly located nucleus. Cytopathogenic effects of parasite development consisted of the formation of holes in the cell monolayer associated with the rupture of infected host cells. Serial passage of tachyzoites was achieved by subinoculation of tachyzoites onto non-infected bovine monocyte cell cultures. It appears that N. caninum can be continuously grown in cell cultures.     

Protracted shedding of oocysts of Neospora caninum by a naturally infected foxhound

Feces from 15 dogs at 2 different foxhound kennels in the U.K. were examined microscopically for the presence of oocysts of Neospora caninum. One sample containing approximately 400 candidate oocysts per gram was positive in a polymerase chain reaction (PCR) using N. caninum-specific primers. In a sample taken 4 mo later from the same hound. N. caninum oocysts were again detected visually and by PCR. This is the third reported case of a dog naturally excreting oocysts of N. caninum and suggests that oocyst excretion can occur over a relatively long period of time in some circumstances or that reshedding may occur.     

Loss of infectivity of Neospora caninum oocysts maintained for a prolonged time

The purpose of this study was to investigate whether sporulated Neospora caninum oocysts, which had been stored for 46 mo in a 2% sulfuric acid solution at 4 degrees C, remain morphologically viable and infective to gerbils (Meriones unguiculatus). Six gerbils were orally inoculated with doses of 400 or 1,200 oocysts. Two mo after inoculation, the animals did not show any clinical signs, had no histological lesions, and were seronegative for N. caninum at 1: 50 in an immunofluorescent antibody test. PCR using the brain from each gerbil did not reveal N. caninum specific DNA. We conclude that oocysts preserved for 46 mo are not infective, despite being morphologically intact.     

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

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

Seroprevalence of Neospora caninum antibodies in dogs in India

Neospora caninum is one of most important causes of abortion in cattle worldwide, and dogs are an important risk factor for N. caninum infection in cattle. Antibodies to N. caninum were determined in 184 (126 rural, 58 urban) dogs from the Punjab State, India, using commercial monoclonal antibody-based competitive ELISA and found in 16.8% of the animals. The prevalence of N. caninum antibodies was significantly higher in rural dogs (21.4%, 27 of 126) than city dogs (6.9%, 4 of 58). To our knowledge this is the first report of N. caninum infection in canines from India.     

Dirofilaria immitis: cell-mediated and humoral immune responses in experimentally-infected dogs.

Four dogs were experimentally infected with 30 Dirofilaria immitis infective larvae, four dogs received two such infections and four dogs served as uninfected controls. A partially-purified D. immitis antigen was used in an indirect hemagglutination assay to determine anti-D. immitis antibody titers. Anti-D. immitis antibody was first detected in infected dogs 4 weeks after infection. Titers were highest 2 weeks after the appearance of microfilariae and diminished to low levels thereafter in the single infection group. Antibody levels in the double infection group decreased similarly but were demonstrable throughout the study. Antibody titers were significantly higher in the infected dogs, but there were no differences in titers between single and double infection groups.The responses of peripheral lymphocytes to phytohemagglutinin P and pokeweed mitogen were significantly depressed in infected dogs. Peripheral blood lymphocyte transformation could not be induced with D. immitis antigens. Differences between groups in T-cell function were not demonstrated by total hemagglutinating antibody or 2-mercaptoethanol labile hemagglutinating antibody following immunization with sheep erythrocytes.     

Prevalence of Neospora caninum infection in dogs from beef-cattle farms, dairy farms, and from urban areas of Argentina

Prevalence of anti-Neospora caninum antibodies was determined in sera of 320 dogs from Argentina using the indirect fluorescent antibody test (IFAT). Antibodies to N. caninum were found in 121 of 320 (37.8%) sera with titers of 1:50 (21 dogs), 1:100 (23 dogs), 1:200 (23 dogs), 1:400 (17 dogs), 1:800 (23 dogs), and > or = 1:1.600 (14 dogs). The seropositivity (IFAT, > or = 1:50) was higher in dogs from dairy (48% of 125) and beef (54.2% of 35) farms than in dogs from urban areas (26.2% of 160). Prevalence of anti-N. caninum antibodies was higher in dogs more than 12 mo of age (47.7%, 105 of 222) versus in 12-mo-old or younger dogs (12.7% of 86), suggesting postnatal exposure of N. caninum infection in dogs.     

Trehalose-6-phosphate Phosphatase from Mycobacterium tuberculosis induces humoral and cellular immune responses

Trehalose-6-phosphate phosphatase is an enzyme strictly essential for the growth of mycobacteria. Subcellular fractionation of Mycobacterium tuberculosis and M. bovis bacillus Calmette-Guérin (BCG) located the trehalose-6-phosphate phosphatase in the cell wall and membrane fractions. Trehalose-6-phosphate phosphatase induced an increased Th1-type immune response in mice, characterized by an elevated level of interferon-gamma in antigen-stimulated splenocyte culture and a strong IgG2a antibody response. The trehalose-6-phosphate phosphatase was recognized by the sera of tuberculosis patients and BCG-vaccinated donors. The mycobacterial trehalose-6-phosphate phosphatase is an immunodominant antigen, and it may be a candidate for vaccine development for the control of tuberculosis.