Abstract
The aim of the study was to diagnose Sarcocystis sp. infections in cattle and to detect coinfections by Toxoplasma gondii and/or Neospora caninum. Blood, diaphragm, esophagus, and myocardium from 90 beef cattle from Argentina were collected. Histopathological, immunohistochemical, polymerase chain reaction assays, and direct microscopical examination were carried out. Sarcocysts from myocardium were measured and counted. Indirect fluorescent antibody test (IFAT) for the three protozoans was performed. Sarcocystis cruzi sarcocysts were found in 100% of myocardium samples. Sarcocysts per gram ranged from 8 to 380 with higher values found in adult cattle (p < 0.001). T. gondii and N. caninum were not detected by immunohistochemistry. T. gondii DNA was found in myocardium of 2/20 seropositive animals, while N. caninum DNA was not found. Antibodies against S. cruzi were detected in all samples, those against N. caninum in 73% and against T. gondii in 91% of the samples (IFAT titer ≥25). It is concluded that serology by IFAT is a suitable method to diagnose these protozoan infections due to its specific IgG detection; therefore, IFAT may be a useful tool to evaluate the impact of each protozoan infection in coinfected animals.
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Introduction
Sarcocystis cruzi, Neospora caninum, and Toxoplasma gondii infections are distributed worldwide in cattle. S. cruzi is the most prevalent of the Sarcocystis species infecting cattle; in many countries, over 90% of adult cattle have been found infected (Dubey et al. 1989). Occasionally, S. cruzi causes abortion, acute systemic illness, and poor growth (Dubey et al. 1989). Reports about Sarcocystis infections in Argentina are scarce, and only the presence of sarcocysts in cattle has been reported (Moriena et al. 1989). N. caninum is now recognized as a major cause of bovine abortions in many countries, including Argentina (Dubey 2003; Venturini et al. 1999). T. gondii infection is zoonotic and usually subclinical in cattle. The role of bovines in the epidemiology of toxoplasmosis is uncertain (Dubey et al. 2005).
Serological methods like indirect fluorescent antibody test (IFAT) and enzyme-linked immunosorbent assays, detect antibodies to S. cruzi, N. caninum, and T. gondii antigens in single or mixed infections (Uggla et al. 1987; Dubey et al. 1996). Qualitative and quantitative diagnosis of Sarcocystis sp. may be accomplished by microscopical examination, but techniques like immunohistochemical staining and polymerase chain reaction (PCR) methods are necessary for the specific diagnosis of N. caninum and T. gondii infection (Dubey and Beattie 1988; Dubey and Schares 2006; Ho et al. 1997).
The aims of the present study were to diagnose Sarcocystis sp. infections, to detect Sarcocystis sp. antibodies by IFAT and to identify coinfections by T. gondii and/or N. caninum in Argentinean beef cattle.
Materials and methods
Cattle
Samples from beef cattle (n = 90) were obtained in two slaughterhouses from Buenos Aires Province, Argentina. Five animals were sampled in each of the 18 visits to the slaughterhouses from June to December 2004 from 3 categories: heifers (n = 30; 14–16 months old), steers (n = 30; 24–30 months old), and adult cattle (n = 30; 10 bulls and 20 cows, 42–112 months old).
There is approximately 50 million head of cattle in Argentina, most of them raised in extensive systems. Sample size was considered representative of the Sarcocystis-infected cattle population based on 90% prevalence with 99% confidence and 10% accuracy (Cannon and Roe 1982).
Samples
Fifteen milliliters of blood, 50 g of diaphragm, 10 cm of esophagus, and 100 g of myocardium (left ventricle) were collected from each animal. Refrigerated samples were transported within 6 h to the laboratory where 1 g of each fresh tissue was processed, 1 g was kept in 10% neutral buffered formalin for histopathological studies, and 1 g was stored at −20°C for molecular assays. Sera obtained from blood samples were stored at −20°C until processing.
Serological tests for S. cruzi, N. caninum, and T. gondii
Sera were tested by the IFAT for the detection of antibodies to S. cruzi, N. caninum, and T. gondii. For S. cruzi antibody detection, bradyzoites from naturally infected bovine hearts were used as antigen. One hundred grams of minced myocardium were mixed with 400 ml of digestion solution (2.5 g pepsin [0.7 U-FIP/mg] and 10 ml HCl in 1 l of water solution) and put in a magnetic stirrer for 20 min at 37°C (Lunde and Fayer 1977, modified). The suspension was filtered through 300, 150, and 53 μm sieves into 50 ml centrifuge tubes and centrifuged at 500×g for 5 min. Supernatants and the upper layer of the sediment were discarded; the pellet was washed three times with phosphate-buffered saline (PBS) and diluted in PBS to a final concentration of 4 million bradyzoites/ml. Bradyzoites were fixed onto 12-well IFAT slides and stored at −20°C. For N. caninum and T. gondii serology, cell culture-derived tachyzoites of NC-1 (Dubey et al. 1988) and RH strains (Sabin 1941), respectively, were used as antigens.
Sera were diluted twofold in PBS starting at 1:25 dilution. A rabbit antibovine IgG fluorescein isothiocyanate conjugate (Sigma Bio Sciences, St. Louis, USA) was used. Bovine positive and negative control sera were used in each analysis. Fetal serum was used as negative control. Complete peripheral fluorescence of merozoites was considered positive. IFAT titers to S. cruzi, N. caninum, and T. gondii were compared by the correlation test.
Cattle sera were also examined for T. gondii antibodies using the modified agglutination test (MAT) as described by Desmonts and Remington (1980) with serum samples diluted as described above.
Microscopical studies
Fresh samples
One gram of fresh myocardium, esophagus, and diaphragm samples from each of the 90 animals were dissected under a stereomicroscope for sarcocysts, and those from the myocardium were counted. Differences in the amount of sarcocysts among cattle categories were evaluated by the goodness of fit test (Epi 6).
Length and wall thickness of 129 sarcocysts from the myocardium (50 from 10 heifers, 27 from 8 steers, and 52 from 12 adults) were measured with a calibrated eyepiece. Differences of cyst length among cattle categories were evaluated by the ANOVA test.
Histopathology and immunohistochemistry
Thirty heart samples that had the greatest counts of sarcocysts or serological titers ≥200 to S. cruzi antigen were selected and embedded in paraffin. For histopathological studies, 5 μm sections were stained with hematoxylin and eosin.
Twenty heart samples from animals with positive IFAT antibody titers for N. caninum (25 to 800) and T. gondii (25 to 100) were processed by immunohistochemical staining for N. caninum and T. gondii with the LSAB+System HRP using a commercial kit according to the manufacturer's instructions (Dako Cytomation, Carpinteria, USA).
Polymerase chain reaction
From each of the 20 samples selected for immunohistochemical staining, 25 to 50 μg of heart was processed for PCR. DNA was isolated using a commercial DNA-extraction kit (DNeasy® Tissue Kit, QIAGEN, Hilden, Germany) according to the manufacturer's recommendations. The amplification was performed with the specific primer pair Np 6/Np21 (Müller et al. 1996; Yamage et al. 1996; Kaufmann et al. 1996) and B22/B23 (Bretágne et al. 1993) for N. caninum and T. gondii, respectively. For N. caninum DNA detection, 1 μl of genomic DNA from each sample was added to tubes with a PCR master mix containing 2.5 μl of 10x PCR buffer (Fermentas, Hanover, USA); 200 μM each of dATP, dTTP, dGTP, and dCTP; 0.15 μl of Taq DNA polymerase (5 U/μl Fermentas); and 0.4 μM from each primer in a final volume of 25 μl. The reaction was accomplished in a thermal cycler (PCR Sprint Thermo Electron Corporation) with an initial denaturation step at 94°C for 5 min, followed by a 35-cycle program with denaturation (94°C; 30 s), annealing (55°C; 30 s), primer extension (72°C; 60 s), and a final extension at 72°C for 7 min. For T. gondii, the same procedure was followed with the exception that the annealing step was performed at 60°C for 30 s. Amplicons were visualized after electrophoresis in a 6% polyacrylamide gel and stained with silver staining (Harlow and Lane 1988). DNA isolated from T. gondii RH strain or N. caninum NC-1 strain were used as positive controls and replaced by nuclease-free water for the negative control.
Results
Microscopical studies
Fresh samples
Sarcocysts were found in 100% of the myocardium, 71% of the esophagus, and 28% of the diaphragm samples. The characteristics of all observed sarcocysts corresponded to S. cruzi: they had thin walls (<1 μm thick) and slender villar protrusions (6–13 μm long). Sarcocysts were 153 to 782 μm long with mean cyst length differing by category (301 μm for heifers, 387 μm for steers, and 442 μm for adult cattle; p < 0.01).
The number of S. cruzi sarcocysts per gram of myocardium ranged from 8 to 380. Figure 1 shows the distribution of sarcocysts per gram of myocardium for each animal category. The total and mean amounts of sarcocysts counted for each category are shown in Table 1. Differences were observed in total amounts of sarcocysts between heifers and cows (p < 0.001), steers and bulls (p < 0.001), and males and females (p < 0.001).
Histopathology and immunohistochemistry
Thin-walled sarcocysts were found in 28 of 30 myocardium sections, while thick-walled sarcocysts were not found. Lesions were not observed. N. caninum and T. gondii tissue cysts or tachyzoites were neither observed in hematoxylin and eosin nor in immunohistochemically stained sections.
Serological tests
Antibodies against S. cruzi antigen were detected in all samples, those against N. caninum in 73% and against T. gondii in 91% of the samples (IFAT titer ≥25). Figure 2 shows the distribution of animals and antibody titers to S. cruzi, N. caninum, and T. gondii antigen. Table 2 shows the frequency of diagnosis of single and mixed infections by IFAT. By MAT, 13 animals were positive to T. gondii antibodies with a titer of 25.
No significant correlation was found between IFAT titers to N. caninum and T. gondii antigen (r = 0.11), titers to S. cruzi and N. caninum antigen (r = 0.15), and titers to S. cruzi and T. gondii antigen (r = 0.13).
Polymerase chain reaction
After electrophoresis, 115 bp amplicons corresponding to T. gondii were detected in DNA samples from a 9-year-old cow and a 2-year-old steer, both of them positive to T. gondii by IFAT (titer of 100). By MAT, the cow had a titer of 25 and the castrated male was negative. The corresponding 328 bp amplicon for N. caninum was not detected in any of the samples.
Discussion
All the sarcocysts observed corresponded to S. cruzi. A thin sarcocyst wall of around 1 μm characterizes S. cruzi and may be observed by fresh microscopical examination; a thick radially striated, 2.5 to 9.0 μm sarcocyst wall characterizes S. hominis and S. hirsuta (Dubey et al. 1989).
The frequency of S. cruzi detection by fresh examination was high, between 28% and 100% in the different tissues. These findings are in agreement with studies from other countries, which reported high prevalence of sarcocystosis by S. cruzi, myocardium being the most frequently infected tissue (Dubey et al. 1989). Previously, Moriena et al. (1989) found sarcocysts in tissues of cattle from north-eastern Argentina in 79 of 100 animals, but the Sarcocystis species was not reported. To our knowledge, this is the first characterization at the species level for S. cruzi in Argentina.
Reports about the count of sarcocysts per gram in cattle tissues were not found in the literature. In the present study, dissection of fresh myocardium allowed the quantification and species identification of sarcocysts. Sarcocysts per gram of myocardium ranged from 8 to 380. The higher proportion of sarcocysts detected in adult cattle suggests that sarcocyst number may increase with age, which could be due to greater opportunities of older animals of consuming sporocysts and/or to the lack of immunological control of reinfections.
Males had proportionally more sarcocysts per gram of myocardium than females. Savini et al. (1992) found a higher prevalence in bulls than in females. They suggested that bulls pastured in close proximity to farm buildings may have increased risk of infection because the interspecies contact is greater in these areas. Similar management practices are applied in Argentina and may be related with higher cyst counts found in males, but differences of immune response between sexes cannot be discarded.
In addition, sarcocysts found in adult cattle were longer than those from young cattle, which indicate that cysts growth continues over time. Sarcocyst counts and measurement by sex and animal category provided novel information on the dynamics of S. cruzi infection.
N. caninum was detected in heart tissue from cattle fetuses by immunohistochemistry and PCR techniques (Dubey and Schares 2006) and by PCR in 1 of 6 experimentally infected cows (Ho et al. 1997). However, we were unable to detect N. caninum by immunohistochemistry or PCR in the myocardium of 20 naturally seropositive animals, which may be due to absence of the parasite in the examined portions or in their hearts.
T. gondii DNA was found in myocardium samples from 2 of 20 seropositive animals, but T. gondii organisms were not detected by immunohistochemical staining. This is the first report of T. gondii DNA detection in Argentinean cattle. The significance of this finding remains to be investigated because isolation was not performed, and therefore, the potential risk of beef as T. gondii infection source could not be assessed. Detection of T. gondii DNA has been reported previously in 3% adult cows, 2% young bulls, 6% heifers, and 1% calves in Switzerland (Wyss et al. 2000).
Antibodies to S. cruzi antigen were detected in all animals with IFAT titers from 25 to 3,200. Titers of 25 are indicative of infection as they could be confirmed with the parasitological findings. To our knowledge, this is the first serological study of Sarcocystis sp. infections in Argentinean cattle. The absence of significant correlation between antibody titers to S. cruzi, N. caninum, and T. gondii antigens suggests that IgG titers ≥25 are specific and animals with positive titers for more than one parasite are coinfected. Further supporting these findings, Uggla et al. (1987) did not detect serological cross reaction in calves experimentally infected with S. cruzi using T. gondii antigen for IFAT at 1:10 dilution, and Dubey et al. (1996) did not detect cross reactivity by IFAT among S. cruzi, N. caninum, and T. gondii in experimentally infected cattle.
Infection by the three protozoans was found in 70% of the animals, by T. gondii and S. cruzi in 21%, by N. caninum and S. cruzi in 3.4%, and only by S. cruzi in 5.6% of animals. More studies are needed to determine if the high coinfection rate also occurs in the larger Argentinean cattle population and determine the potential implications of coinfection with these parasites on beef production.
In conclusion, serology by IFAT is a suitable method to diagnose S. cruzi, T. gondii, and N. caninum infections in cattle due to its specific IgG detection; therefore, IFAT may be a useful tool to evaluate the impact of each protozoan infection in coinfected animals. In addition, myocardium samples for PCR and IHQ seem to be less appropriate to diagnose N. caninum and T. gondii infections in adult cattle.
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Acknowledgements
We would like to thank Dr. J. Idiart for the assistance in the histopathological studies, Dr. M. Risso for the helpful suggestions about the statistical analysis, Dr. M. Rambeaud for her critical reviewing of this manuscript, Dr. E. Pennimpede for his valuable help, and Mr. I. Ercoli for the technical assistance.
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Financial support for this study was provided by SeCyT through BID 1728 PICT No. 10858/8.
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Moré, G., Basso, W., Bacigalupe, D. et al. Diagnosis of Sarcocystis cruzi, Neospora caninum, and Toxoplasma gondii infections in cattle. Parasitol Res 102, 671–675 (2008). https://doi.org/10.1007/s00436-007-0810-6
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DOI: https://doi.org/10.1007/s00436-007-0810-6