Frequency of Neospora caninum infections in beef cow-calf operations under extensive management.

The aim of this study was to evaluate the frequencies of Neospora caninum horizontal and vertical transmissions in beef cow-calf operations under three different extensive management systems: group A: 0.75 head per hectare pasturing on natural grass; group B: 1.1 head per hectare on natural grass and improved cultured pastures; and group C: 2 head per hectare on natural grass, improved cultured pasture and whole corn silage. Serum samples from 72 multiparous cows assigned to each beef cow-calf operations were obtained every 3 months during 2 years. A group of 30 replacement heifers from each group were tested similarly since they were 10-21 months old. Twenty four, 20 and 34 calves from groups A, B and C respectively, were bled before colostrum intake and again 6 months later. The samples were analyzed by indirect fluorescence antibody test (IFAT) for detection of total IgG against N. caninum at a serological titre ≥ 200 for multiparous cows and replacement heifers, and a serological titre ≥ 25 for calves. Serum samples from seropositive cows were assessed by ELISA to evaluate the avidity of their specific antibodies. There were no differences in the proportion of seropositive cows from groups A, B and C at the beginning of the trial (p>0.05). Interestingly, the lowest serological titres in seropositive cows from all groups were observed during the first trimester (p<0.05). Although seropositive cows had medium to high avidity antibodies, suggesting chronic infection; seroconversion associated with low antibody avidity was found in 2, 3 and 3 seropositive cows from groups A, B and C. All replacement heifers remained seronegative. No abortions were recorded but 2, 1, and 2 calves from groups A, B and C were seropositive before colostrum intake, respectively. Seropositive calves born from cows having intermediate or high avidity remained with the same serostatus at 6 months of age. Even under varying extensive management conditions, both N. caninum horizontal and vertical transmission methods do occur in beef cow-calf operations.

Neospora caninum serostatus is affected by age and species variables in cohabiting water buffaloes and beef cattle.

The aim of this study was to investigate how Neospora caninum serostatus may be affected by variables such as host species (water buffaloes or cattle) and age in animals cohabiting in the same ranch. A convenience cross-sectional study was performed on four ranches in the Northeast of Argentina, where water buffalo are cohabitating with beef cattle. Blood samples were collected from 1350 female water buffaloes (Bubalus bubalis) and 880 female beef cattle (Bos taurus and Bos indicus crossbreeds) from four ranches. Calving and weaning percentages at herd level for each ranch were also recorded. N. caninum antibody levels were measured by an indirect fluorescent antibody test (IFAT) (reciprocal antibody titers ≥ 100). Serological results were classified into 2 categories (0: negative; 1: positive). A logistic regression model was used to describe the relationship between N. caninum serostatus and specie (water buffalo or cattle), age or ranch and their interactions. Likelihood ratio tests were used to assess the significance of the model and their terms. Odds ratios were estimated and 95% profile likelihood (LR) and Wald confidence intervals (CI) obtained. Overall, specific antibody titers were found in 43.3% (584/1350) of water buffaloes and 28.6% (252/880) of cattle. Seropositive water buffaloes and cattle were observed on all ranches. Age was statistically significant (p=0.01) with an overall estimate of logit (log odds) of age of 0.03 for both species. This indicates that for every one year increase in age, the expected change in log odds of being seropositive increased by 0.03. On three of four ranches a water buffalo was 4.48, 1.54 and 2.25 times more likely to be seropositive than cattle for animals of the same age. The N. caninum serostatus was affected by age in the first place, but also by species on at least three of the four ranches. Calving and weaning percentages were higher in water buffaloes than in beef cattle (p<0.05). Even though the low pathogenicity that N. caninum seems to have in water buffaloes, this study reinforces the importance of this specie as maintenance of the disease.

The Neospora caninum-Spain 7 isolate induces placental damage, fetal death and abortion in cattle when inoculated in early gestation.

The Nc-Spain 7 isolate of Neospora caninum, which was newly obtained from an asymptomatic congenitally infected calf, demonstrated a similar virulence as Nc-1 strain in mouse models. The aim of this study was to characterize the pathogenesis of Nc-Spain 7 isolate in cattle after experimental infection at 65 days of gestation. For this purpose, thirteen pregnant heifers were divided into three groups as follows: group A: 7 heifers inoculated with 1 × 10(8) tachyzoites of Nc-Spain 7 isolate; group B: 4 heifers inoculated with 1 × 10(8) tachyzoites of Nc 1 strain; and group C: 2 heifers received PBS. Serum samples were collected weekly and heparinized blood samples were collected three times (0, 28 and 42 days after inoculation) by jugular venipuncture. Placenta and fetal tissue samples were collected at time of necropsy. Specific antibody response in the dams was tested by IFAT, indirect ELISA, and rNcGRA7 and rNcSAG4 based-ELISA. Specific antibody response in fetal fluids was tested by IFAT. IFN-γ production was measured after in vitro culture of PBMC and the supernatant was assessed using a commercial kit (BOVIGAM). A significant increase in N. caninum antibody responses was detected in groups A and B by IFAT and by i-ELISA from day 14 after inoculation onwards. Besides, antibody response against rNCGra7 protein was also detected in all inoculated heifers by rNcGra7-based ELISA. Four fetuses from group A and one from group B were aborted between 3 and 5 weeks after infection. In the recovered fetuses, only 3 out of 4 fetal fluids from fetuses of group A and 1 out of 3 of group B were seropositive by IFAT, but all of them were positive by PCR. Transplacental transmission could be determined in all fetuses from groups A and B by PCR and/or IHC. Heifers of group C and their fetuses remained negative by all techniques. The results of this study demonstrate that the NC-Spain 7 isolate could be transmitted transplacentally, and produced fetal death and abortion in cattle.

Experimental inoculation of Neospora caninum in pregnant water buffalo.

The aim of this study was to characterize the pathogenesis of Neospora caninum in experimentally inoculated pregnant water buffalo (Bubalus bubalis). Twelve Mediterranean female water buffaloes ranging in age from 4 to 14 years old and seronegative to N. caninum by indirect fluorescent antibody test (IFAT) were involved. Ten females were intravenously inoculated with 10(8) tachyzoites of NC-1 strain at 70 (n=3) or 90 (n=7) days of pregnancy (dp). Two control animals were inoculated with placebo at 70 and 90 dp, respectively. Serum samples were obtained weekly following inoculation to the end of the experiment. Three animals inoculated at 70 dp were slaughtered at 28 days post inoculation (dpi), three animals inoculated at 90 dp were slaughtered at 28 dpi and the remaining four animals inoculated at 90 dp were slaughtered at 42 dpi. Fetal fluids from cavities and tissue samples were recovered for IFAT and histopathology, immunohistochemistry and PCR, respectively. Genomic DNA from fetal tissues was used for parasite DNA detection and microsatellite genotyping in order to confirm the NC-1 specific-infection. Dams developed specific antibodies one week after the inoculation and serological titers did not decrease significantly to the end of the experiment. No abortions were recorded during the experimental time; however, one fetus from a dam inoculated at 70 dp was not viable at necropsy. Specific antibodies were detected in only two fetuses from dams inoculated at 90 dp that were slaughtered at 42 dpi. No macroscopic changes in the placentas and organs of viable fetuses were observed. Nonsuppurative placentitis was a common microscopic observation in Neospora-inoculated specimens. Microscopic fetal lesions included nonsuppurative peribronchiolar interstitial pneumonia, epicarditis and myocarditis, interstitial nephritis, myositis and periportal hepatitis. Positive IHC results were obtained in two fetuses from dams inoculated at 70 dp and slaughtered at 28 dpi. N. caninum DNA was detected in placentas and fetuses from all inoculated animals. The pattern of amplified microsatellites from placental and fetal tissues resembled the NC-1 strain. Water buffaloes, like cattle, are susceptible to experimental inoculation with N. caninum at early pregnancy.

The role of Neospora caninum and Toxoplasma gondii in spontaneous bovine abortion in Argentina.

The aim of the present work was to evaluate the role of Neospora caninum and Toxoplasma gondii infections in spontaneous bovine abortions in Argentina. Based on histopathological results, 70 presumptive cases of apicomplexan protozoal abortion from a total of 666 cases of spontaneous bovine abortion submitted to the National Institute of Agrarian Technology, Balcarce, from 1999 to 2007 were included in this study. N. caninum infection was diagnosed by an indirect fluorescent antibody test (IFAT), by immunohistochemistry (IHC) and by nested-PCR. T. gondii infection also was diagnosed by nested-PCR. DNA from fetuses was extracted primarily from CNS tissues. Heart, liver, muscle and/or placenta were processed when nervous tissue was not available. Sixty-six (9.9%) fetuses were positive by at least one technique (IFAT, IHC or nested-PCR) for N. caninum infection. Overall, there was poor agreement among results obtained by these diagnostic techniques. In contrast, no Toxoplasma-infection was detected in any aborted bovine fetus.

Humoral immune response in pregnant heifers inoculated with Neospora caninum tachyzoites by conjunctival route.

The aim of this study was to compare systemic humoral immune responses in pregnant heifers inoculated with Neospora caninum tachyzoites by conjunctival and intravenous routes. Twenty nine heifers separated in three experimental groups were studied: Group 1 (n=10 animals) and Group 2 (n=9 animals) were inoculated with 10(8) of N. caninum tachyzoites by conjunctival and intravenous routes at 5th month of gestation, respectively; Group 3 (n=10 animals) were non-inoculated control animals. An indirect fluorescent antibody test (IFAT) and western immunoblotting (IB) were used to analyze the humoral immune response. All animals from Group 1 developed N. caninum specific antibody responses after conjunctival inoculation recording the highest antibody titer (mean+/-SE: 160+/-49.9) at 6th month of gestation. There were statistical differences between humoral immune responses found in Group 1 and 2 being higher in the second one at 6.5th, 8.5th and 9th months of gestation (P<0.05). Interestingly, all heifers from Group 1 reverted to seronegative status at the end of gestation. No increase in antibody was detected in the uninfected control group. Same pattern of N. caninum antigens was recognized by sera from heifers inoculated by conjunctival route and heifers inoculated by intravenous route. Recognized antigens were 116, 92, 84, 77, 45, 40, 25-26 and 17-18 kDa. The conjunctival instillation of N. caninum tachyzoites in pregnant heifers induces specific systemic antibodies. Further work is needed in order to clarify the consequences of this novel experimental route of infection not only on the fetus but also on the dam.

Excimer laser lamellar keratoplasty.

PURPOSE: To evaluate a procedure using the excimer laser to perform lamellar keratoplasty to treat deep corneal scars in the central optical zone. To determine if excimer laser can safely prepare a smooth surface for the host bed and the donor button, assess the interface opacity and evaluate the effects of the ablation on the recipient's endothelial surface with the deeper ablation. METHODS: Nineteen rabbits underwent an excimer laser lamellar keratoplasty in one eye. The rabbits were followed for 9 to 12 weeks until they were sacrificed. RESULTS: Little opacity developed at the graft-host interface and scanning electron microscopy of the endothelial surface showed little difference between the treated and untreated areas or the endothelium of the untreated eyes. CONCLUSIONS: Our results suggest that the use of the excimer laser to treat corneas with deep stromal scars and normal endothelium is feasible. However, we have not proved that this technique will give a better result than conventional lamellar dissection methods.

The effect of hEGF and insulin on corneal metabolism during Optisol storage.

We examined the effect of the growth factors, human epidermal growth factor (hEGF) and insulin, on corneal metabolism during storage in Optisol, a chondroitin-sulfate-(CS)-based storage medium. Paired cat corneas, in either Optisol only or Optisol with growth factor(s), were analyzed using ex vivo 31P nuclear magnetic resonance, after storage for 1 week at 4 degrees C. Lysosomal enzyme release into the media at the end of the storage period also was measured fluorometrically. Both epithelial-intact and epithelial-denuded corneal pairs were examined for all conditions. Considering corneas having either intact epithelia or epithelium-denuded corneas, the addition of either growth factor alone to Optisol did not alter the relative corneal concentrations of five of the six phosphatic metabolite spectral bands measured or two metabolic ratios calculated from these bands. Phosphodiesters, however, were significantly lower in corneas stored in Optisol containing both hEGF and insulin (23%) than in corneas stored in Optisol alone (30%). Intracorneal pH was unaffected by the addition of growth factor(s). A significantly higher release of alpha-glucosidase and alpha-mannosidase was noted in those corneas stored in Optisol containing both hEGF and insulin. Optisol maintains high-energy phosphate corneal metabolism similar to other CS-based media, K-Sol and Chondroitin Sulfate Corneal Storage Medium (CSM). The addition of the growth factors hEGF and insulin to Optisol alters corneal metabolic activity during storage in a manner indicative of conserving corneal phospholipids.

A randomized, prospective, double-masked clinical trial of Optisol vs DexSol corneal storage media.

We compared Optisol and DexSol, two chondroitin-sulfate-based media for corneal storage at 4 degrees C, by transplanting 31 donor cornea pairs (one cornea stored in Optisol and its mate in DexSol for 20 to 134 hours) into 31 pairs of recipients (62 patients). All grafts were clear 1 year after transplantation except for one primary donor failure (Optisol group). Optisol-stored corneas were significantly thinner than DexSol-stored corneas after cardinal suture placement (0.64 mm vs 0.76 mm) and at the end of surgery (0.69 mm vs 0.78 mm); at all points afterward through 1 year the two groups did not differ. The activity of two lysosomal enzymes released into the media during storage, alpha-mannosidase and alpha-glucosidase, was lower in the Optisol group. Epithelial status and all endothelial morphometric parameters, except the figure coefficient at 1 year, did not differ between the two groups before surgery and 3, 6, and 12 months after surgery. Mean endothelial cell loss at 1 year was 15% for the Optisol group and 21% for the DexSol group (P = .22). Thus, Optisol-stored corneas were thinner during surgery than DexSol-stored corneas and there was less lysosomal enzyme activity in the Optisol medium after tissue storage. There were no significant differences in postoperative clinical or endothelial morphometric parameters, however.

A donor-age-dependent change in the activity of alpha-mannosidase in human cultured RPE cells.

Six acidic glycosidase activities in cultured human retinal pigment epithelium (RPE) cells from donors of different ages (19 to 80 years) were studied with regard to pH optimum, Km, Vmax and specific activity. For alpha-mannosidase we found significant age-dependent decreases in specific activity and Vmax but not in Km. The other glycosidases and acid phosphatase, lactate dehydrogenase (LDH) and citrate synthase showed no change in these parameters with donor age. The alpha mannosidase activity of older donor cells could be activated almost 2-fold by the addition of zinc. This is the first report of age-dependent change in a human RPE lysosomal enzyme. Since alpha-mannosidase is probably required for the degradation of rhodopsin in the phagolysosomal system of the RPE, decrease in this enzyme activity may lead to accumulation of undigested rod outer segments (ROS) and drusen, both of which are associated with age-related macular degeneration (AMD).