Parasitol Res DOI 10.1007/s00436-010-2146-x
ORIGINAL PAPER
Neospora caninum: evaluation of vertical transmission in slaughtered beef cows (Bos indicus) F. A. C. Marques & A. S. Headley & V. Figueredo-Pereira & A. Taroda & L. D. Barros & I. A. L. Cunha & K. Munhoz & F. M. Bugni & D. L. Zulpo & M. Igarashi & O. Vidotto & J. S. Guimarães Junior & João Luis Garcia
Received: 20 August 2010 / Accepted: 28 October 2010 # Springer-Verlag 2010
Abstract Neospora caninum is a protozoan parasite that causes the most important reproductive problems in cattle worldwide. The objective of this study was to evaluate the possibility of vertical transmission of N. caninum in zebus breed beef cows (Bos indicus) submitted for slaughter at an abattoir in the northern region of the State of Paraná, southern Brazil. One hundred and fifty-nine cows were evaluated: 83 pregnant (in different stages of gestation) and 76 non-pregnant. Serum determination of N. caninum was evaluated by indirect ELISA (Idexx). Blood (with EDTA) from pregnant cows and tissue samples (brain and heart) from their fetuses were collected and used for PCR analyses. Antibodies against N. caninum were observed in 14.6% (12/83) of pregnant and in 15.8% (12/76) of nonpregnant cows. Antibodies against the parasites were detected in one fetus (1.4%). The PCR analyses revealed that 6.0% (5/83) of cows and 4.8% (4/83) of fetuses evaluated were positive to specific N. caninum primers. These positive fetuses were between 4 and 6 months of age. Thus, considering PCR and serology as an indicative of vertical transmission in fetuses, 4.8% of fetuses were infected by N. caninum during gestation.
F. A. C. Marques : V. Figueredo-Pereira : A. Taroda : L. D. Barros : I. A. L. Cunha : K. Munhoz : F. M. Bugni : D. L. Zulpo : M. Igarashi : O. Vidotto : J. S. G. Junior : J. L. Garcia (*) Departamento de Medicina Veterinária Preventiva, Universidade Estadual de Londrina—UEL, Postal Box 6001, 86050-970, Londrina, PR, Brazil e-mail:
[email protected] A. S. Headley Department of Basic Veterinary Sciences, School of Veterinary Medicine, St. Matthew’s University, Grand Cayman, Cayman Islands
Introduction Neospora caninum is a protozoan parasite that is morphologically similar to Toxoplasma gondii. Its life cycle was determined in 1998 (McAllister et al. 1998); dogs and coyotes (Canis latrans) are presently described as definitive hosts (McAllister et al. 1998; Gondim et al. 2004). Several productive and species of wild animals have been identified as the intermediate hosts of N. caninum (Dubey and Lindsay 1996). In dairy herds, transmission is principally by the vertical route (Schares et al. 1998). Calves might be born seronegative, and later become infected by the ingestion of water and food contaminated by oocysts, thereby demonstrating horizontal transmission in the epidemiological chain of the coccidia (Mcallister et al. 1998; Wouda et al. 1999; Anderson et al. 2000). Although several studies relative to the vertical transmission of dairy cattle have been done, there is not adequate documentation of this phenomenon in beef herd. Thus, this study evaluated the possibility of vertical transmission of N. caninum in the zebus breed of beef cows slaughtered in an abattoir located in the northern region of the state of Paraná, Brazil.
Material and methods Study area and sampling This study was carried out in an abattoir located in the city of Londrina, Southern Brazil. One hundred and fifty-nine samples were collected weekly from beef cows (Bos indicus) submitted for slaughter during January to July of 2007; 83 cows were in different stages of gestation, 76 were not pregnant. The pregnant animals were selected at the
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slaughter line in such a manner so that for each pregnant animal, a non-gravid cow with similar characteristics (origin and age), was chosen. This study was approved by The Animal Ethics Committee, Universidade Estadual de Londrina (No 088/2007). Anti-N. caninum antibody detection Antibodies against N. caninum were detected by HerdCheck N. caninum Antibody Test Kit (Idexx laboratories). Sera from the cows were diluted at the ratio of 1:100, and fetuses at 1:25. Blood and tissue samples Blood samples with EDTA were collected from the pregnant cows after they were bled along the inspection line and from their fetuses by cardiac puncture. The white blood cells were separated by centrifugation (550×g, 10 min), and used for DNA extraction and subsequent PCR analyses. Approximately 10 g of tissue fragments (brain and myocardium) were collected for fetuses ≥3 months of age, while similar tissue fragments (10 g) for fetuses ≤3 months were pooled for DNA extraction. The tissue samples were homogenized with 10 ml of saline solution (0.14 M NaCl), after which the homogenate was filtered, and 2 ml were used for DNA extraction. DNA extraction and PCR The DNA extraction and PCR analyses were performed as described (Garcia et al. 2006). Briefly, the blood samples were stored at −20°C prior to extraction. The homogenized sample (250 μl) was transferred to a microtube containing an equal volume of extraction buffer (200 mM NaCl, 20 mM Tris, 50 mM EDTA, proteinase K 1 mg/ml, and 2% SDS), and then incubated at 56°C for 1 h. Thereafter, buffered phenol (250 μl) was added to the mixture, which was then centrifuged at 13,000×g for 5 min. The resulting aqueous phase was transferred to another microtube containing phenol/chloroform/isoamyl alcohol, and centrifuged at 13,000×g for 5 min. DNA precipitation using sodium acetate and ethanol was performed as previously described (Sambrook et al. 1989). The amplification of N. caninum DNA was done by using the Np21 and Np6 primers (Muller et al. 1996). The PCR cycle was performed in a mixture (final volume of 25 μl) containing 5 μl of extracted DNA admixed with 20 μl of a solution containing 1.0 mM of each primer, 100 mM dNTP (Invitrogen, Life Technologies, USA), 60 mM Tris–HCl (pH 9.0), 15 mM (NH4)2SO4, 1.5 mM MgCl2, and 0.5U Taq DNA polymerase (Invitrogen, Life Technologies, USA). The amplification of parasitic genomic DNA was done during 35 cycles in a
thermocycler PTC-100 (MJ-Research), using the following cycling conditions: 5 min at 94°C, followed by 35 cycles of 30 s at 94°C, 30 s at 65°C, and 60 s at 72°C; the 35th cycle was followed by a final extension of 7 min at 72°C. Aliquots of each PCR were visualized by electrophorese on 2% agarose gel. For positive controls, the DNA extracted from tachyzoites of the NC-1 strain (106/ml) of N. caninum, diluted in TE buffer, were used. The negative control consisted of commercially prepared water samples without N. caninum. A positive and negative control was included in each assay. Statistical analysis All variables were analyzed by the Chi-square test (χ2) corrected by Yates and the Fisher Exact Test using the Epi Info program (CDC, 6.04b version). The association between variables and the occurrence of seropositivity were estimated from values obtained by the odds ratio (OR), with a confidence interval of 95%. A P-value of≤0.05 was considered as significant.
Results Seroprevalence of N. caninum The serologic results for N. caninum are given in Table 1. From a total of 159 cows, 24 (15.1%) reacted positively to N. caninum. Positive seroreactivity to N. caninum occurred in 14.4% (12/83) of the pregnant cows, while non-gravid animals revealed a positivity of 15.8% (12/76); however, these values were not statistically different (OR=0.90, 0.354.5 6(14.3) Trimester of gestation First 2(10.5) Second 8(15.4) Third 2(16.6)
Vertical transmission During this study, vertical transmission was demonstrated in 4.8% (4/83) of fetuses by PCR. The DNA extracted from the brain and myocardial samples derived from the fetus (4.5 months of age) originated from cow No. 89 revealed positive results by PCR (Table 2). Cerebral-derived DNA from the fetus (4 months old) of cow No. 97 and the fetus (6 months old) from cow No. 148 was positive by PCR analysis. Myocardial-derived DNA of the fetus (5 months old) originated from cow F149 revealed positive by PCR reaction (Table 2). The DNA-derived blood samples from five cows (animals No. 13, 15, 26, 146, and 147) were PCR Table 2 The outcome of N. caninum infection in slaughtered beef cows (B. indicus) and their fetuses
N, negative; +, positive a
Optical density from ELISA (Idexx)
Cattle No.
ELISAa Cow
Negative
P
OR
IC (95%) OR
0.98a
0.90
0.35–2.33
71 64 141
83 76 159
78 20 37
91 26 42
0.43a
17 44 10
19 52 12
0.85b
positive, but only one animal (No. 26) had a corresponding serum titer (OD=0.93). However, the fetuses of these animals was neither serologically or PCR positive. The Np21 and Np6 primers were able to detect dilutions equivalent of 10−1 tachyzoites/ml (Fig. 1).
Discussion To the best of the authors' knowledge, this is the first study that investigated the possibility of vertical transmission of N. caninum in B. indicus. Herein, the PCR results have demonstrated that 4.8% of the fetuses evaluated were Age of fetus (months)
Fetus
PCR Cows Blood
Brain
Fetuses Myocardium
13 15 26 52
N N 0.93 1.9
N N N N
1 2 3 4
+ + + N
N N N N
N N N N
54 74 89 91 97 103 146 147 148 149 157 159 161
1.5 0.72 1.72 1.7 N 2.13 N N 2.9 1.75 1.52 0.53 0.67
N N N N N N N N 1.45 N N N N
5 4 4.5 7 4 3 5 4 6 5 7 4 6
N N N N N N + + N N N N N
N N + N + N N N + N N N N
N N + N N N N N N + N N N
Parasitol Res Fig. 1 Sensitivity determinations of N. caninum Np21 and Np6 primers. Lane 1, M-100 bp mol. wt marker (Gibco BRL); lanes 2–9, dilutions of tachyzoites; lane 10, negative control
PM
104
103
infected by N. caninum, and only one animal had corresponding antibody titers. Moore et al. (2002) did not observe antibodies by IFAT in six fetuses aborted from seropositive dams, and suggested that this might have been due to an inadequate fetal humoral response. During this study, the age of the fetuses that were positive by PCR varied between 4 and 6 months. Under natural conditions, most infections caused by N. caninum are “in utero”, and considering that the immune system of the fetus is in development, this parasitic-induced reaction can be classified as self (as is observed in some viral infections) without producing an immune response. Similar results have been described (Williams and Trees 2006), where fetuses might develop some form of immunological tolerance, which permits persistence and repeated recrudescence of past infection. However, different results were observed when 11 aborted fetuses without histopathological lesion consistent with that of neosporosis demonstrated positive reactivity by IFA at a dilution of 1:25 (Moore et al. 2002). Vertical transmission has been described as the major factor responsible for the maintenance of this parasite within a cattle herd (Paré et al. 1996; Davison et al. 1999; Innes et al. 2002). After being infected by N. caninum, a pregnant cow might have three possible outcomes: (1) abortion, (2) give birth to a weak calf, or (3) give birth to a clinically health but persistently infected calf. Additionally, congenitally infected heifers can transmit the parasite to their own offspring (Innes et al. 2002). Another important consideration is the fact that different populations of animals (for example: Bos taurus x B. indicus) might also demonstrate variations in their susceptibility to infection (Innes et al. 2002). Further, Aguiar et al. (2006) have described the occurrence of higher seroprevalence in beef farms relative to dairy or mixed farms. This phenomenon might represent the manifestation associated with each management system, i.e., the differences between having or not access to infectious sources, the nutritional differences and emphasis of production activity (such as milking, weaning), or the variability in the duration of time during which the animals are maintained within breeding programs. However, breed predisposition for N. caninum infection
102
10
1
10-1
10-2
10-3
NC
cannot be excluded and should be further investigated (Hornok et al. 2006). The prevalence of N. caninum in infected beef cattle observed during this study was similar to previous descriptions (Moore et al. 2002; Bañales et al. 2006; Armengol et al. 2007; Moore et al. 2009), but was lower relative to other reports (Osawa et al. 2002; Aguiar et al. 2006). The prevalence of N. caninum in dairy and meat cattle from Paraná state (the region where this study was done) was previously described as ranging from 14.31% to 34.8% (Locatelli-Dittrich et al. 2001; Ragozo et al. 2003; Guimarães et al. 2004; Locatelli-Dittrich et al. 2008), and 26.7% (Ragozo et al. 2003), respectively. Therefore, caution should be taken when the results of prevalence studies are being evaluated, since the differences in results might be directly related to the serological techniques employed, the cut-off values, sample size, and the type (breed and/or species) of animal that is being investigated. During this study, the serum prevalence of Neospora infection did not increase proportionally with the age of the infected animal; suggesting that vertical transmission was important for the development of disease in the animals studied. Further, if horizontal transmission was an important epidemiological route of N. caninum-induced infections, the seroprevalence in cows would have been comparatively more elevated in heifers due to the possibility of the animal becoming more exposed to the parasite with increasing age (Bañales et al. 2006). We did not observe a statistical difference between seropositive pregnant (14.4%) and non-pregnant cows (15.8%) during this study. Both cellular and humoral immune responses are responsible for the control of N. caninum. The first mechanism is considered as the primary immune response; however, antibodies can possibly reduce the dissemination of extracellular tachyzoites by neutralizing cell infection. During pregnancy, there are significant changes to the immune response of the host, which might influence the pathogenesis of this disease. Lymphocyte proliferation and IFN-γ down-regulation have been described during mid-gestation (Innes et al. 2001). The events that occur during pregnancy and the influences of pre-natal
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exposure to the parasite on the development of an immune response are critical issues, which have to be addressed (Williams and Trees 2006), if the associated mechanism is to be completely understood. During this study, the presence of antibodies against N. caninum in cows did not correlate exactly with the PCR results; since only one animal (No. 26) demonstrated positive reaction by both diagnostic methods. However, three out of four fetuses that demonstrated PCR-positive results were borne from serologically positive dams. Additionally, one of these fetuses (derived from cow No. 148) revealed an elevated antibody titer, and its dam demonstrated the most elevated antibody response. It was recently demonstrated that the vast majority of cows that undergo N. caninum-induced abortion has higher antibody levels when compared to their non-aborting counterparts (Dubey and Schares 2006). However, McInnes et al. (2006) did not describe a correlation between the DNA and antibodies of N. caninum in dams, fetuses, or abortion due to N. caninum, and have suggested that the presence or absence of antibodies or DNA of N. caninum does not support nor exclude the participation of this parasite in the etiopathogenesis of bovine abortion. In conclusion, this study has successfully demonstrated the first occurrence of vertical transmission (4.8%) by N. caninum in pregnant zebu beef cows from Brazil. Acknowledgment
J.L. Garcia is recipient of CNPq fellowship.
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