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Introduction
Neospora caninum is an Apicomplexa identified as a causal agent of reproductive problems in cattle worldwide (DUBEY, 2003). In their life cycle, dogs (MCALLISTER et al., 1998), coyotes (GONDIM et al., 2004b), and grey wolves (DUBEY et al., 2011) are definitive hosts. N. caninum easily survives in many mammals, identifying multiple intermediate hosts such as cows, sheep, goats, deer (CABRAL et al., 2009; PANADERO et al., 2010; SALABERRY et al. 2010; VARASCHIN et al., 2012), and alpacas (CHÁVEZ-VELÁSQUEZ et al., 2004).
N. caninum is a major cause of spontaneous abortion in bovines and is influenced by several factors. Some reports showed that seropositive cows are three to seven times more likely to abort than seronegative cows (HALL et al., 2005; MOEN et al., 1998). In low-seroprevalence herds (<5%), spontaneous abortions due to N. caninum infection might occur at a rate of 1 per 100 cows/year. In high-seroprevalence herds (>20%), abortions are frequent all year round (THURMOND & HIETALA, 1997). Therefore, seropositivity in herds might increase with age or gestation number, and horizontal transmission can influence this event (JENSEN et al., 1999; RINALDI et al., 2005). Based on N. caninum seropositive herd risk, control of neosporosis in cattle is necessary to prevent reproductive problems outbreaks.
The valley of Lima is one of livestock regions with the highest milk production from Peru; nevertheless, cattle suffer from constant reproductive diseases, principally aborts (GAMARRA, 2001; RIVERA, 2001). Several studies have found N. caninum high prevalence in dairy cattle from Lima Valley (GAMARRA et al., 2009; SILVA et al., 2002). Considering N. caninum presence such a risk in reproductive cattle health (DUBEY et al., 2007), the identification of N. caninum in aborted fetus in areas with previous high reports of neosporosis is essential.
Several diagnostic assays have been employed with varying degrees of success to identify N. caninum. Serology diagnostic assays are the most frequently used given their simplicity and speed; nevertheless, their diagnostic capacity depends on the cutoff value for the assay, as well as on their cross-reactivity with other parasites (ORTEGA-MORA et al., 2006) or their ability to be influenced by pregnancy (NOGAREDA et al., 2007). Polymerase chain reaction (PCR) is the most suitable diagnostic assay, but is expensive and different target genes might influence its sensibility (AL QASSAB et al., 2010; ELLIS, 1998). Histopathology has a low sensitivity for detection when compared to other diagnostic tests; however, it still holds important diagnostic value (DUBEY & SCHARES, 2006; SÁNCHEZ-CASTILLEJA & RODRÍGUEZ-DIEGO, 2018). Overall, the diagnosis of neosporosis is difficult, and more than one diagnostic test should be used to make a correct diagnosis.
In this context, the present study aimed to identify N. caninum seroprevalence and risk factors in dairy cattle. In addition, fetuses aborted of these cows were applied multiple diagnostic assays to demonstrate and understand the impact of neosporosis in reproductive losses in Peru.
Materials and Methods
Samples
The present study was developed according to the Animal Use Ethics Committee from Universidad Peruana Cayetano Heredia, Lima- Peru (protocol number 64237).
A total of 219 serum samples from dairy cattle with records of spontaneous abortion were collected to detect antibodies against N. caninum. All bovines were of Holstein breed and had a semi-intensive-to-intensive breeding system. Additionally, 68 aborted bovine fetuses at different stages of gestation from 7 milk farms were collected to detect the presence of N. caninum (Table 1). The samples collected from the fetuses were intrathoracic liquid, as well as the brain, lung, liver, adrenal gland, heart, kidney, thymus, spleen, bone marrow, and intestine. The intrathoracic liquid was obtained by direct puncture; fetal organs were obtained by necropsy and stored in 10% formaldehyde. Blood samples were obtained by direct puncture of the jugular vein in collection tubes without anticoagulant; waited for coagulation and centrifuged for 1.600g for 10 minutes at room temperature. Foetus age was calculated based on reproductive registers.
Table 1 PCR, ELISA and Histologic assay from fetuses aborted recollected of dairy cattle in Lima Valley, with at least one positive test.
|
N° Farm |
No. Fetuses | ELISA | PCR | Histopathologic lesions according tissues recollected | |||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| BRA | LUN | LIV | KID | HEA | TIM | SPL | BM | AG | INT | Autolysis (%) | |||||
| 1 | 1 | - | + | - | - | AU | - | - | - | - | AU | - | AU | 20 | |
| 1 | 2 | + | + | + | + | + | - | + | - | - | NR | - | NR | - | |
| 1 | 3 | + | + | + | - | - | - | - | - | - | AU | - | - | 10 | |
| 2 | 4 | + | + | - | - | - | - | - | - | - | AU | - | NR | 10 | |
| 4 | 5 | + | + | - | - | AU | - | - | - | - | AU | - | - | 20 | |
| 4 | 6 | + | + | + | - | - | - | + | - | - | NR | + | NR | - | |
| 4 | 7 | - | + | - | - | - | - | AU | - | - | AU | - | - | 20 | |
| 5 | 8 | + | + | + | - | + | - | + | - | - | AU | - | NR | 10 | |
| 5 | 9 | + | + | + | - | - | - | + | + | - | - | - | NR | - | |
| 6 | 10 | + | + | - | - | AU | - | - | - | - | AU | - | - | 20 | |
| 6 | 11 | + | + | - | - | - | - | - | - | - | NR | - | AU | 10 | |
| Total | 5/11 | 1/11 | 2/11 | 1/11 | 4/11 | 1/11 | 0/11 | 0/11 | 1/11 | 0/11 | 8/11 | ||||
| % | 45.4 | 9.1 | 18.2 | 9.1 | 36.4 | 9.1 | 0 | 0 | 9.1 | 0 | 72.7 | ||||
+ Histologic lesion compatible with N. caninum; -: No histological lesion compatible with N.caninum; BRA: brain; LUN: Lung; LIV: Liver; KID: Kidney; HEA: Heart; TIM: Thymus; SPL: Spleen; BM: Bone marrow; AG: Adrenal gland; INT: Intestine; AU: Autolysis; NR: No recollected. PCR: Polymerase chain reaction.
Samples were collected from June 2015 to May 2016 in different milk farms from Lima Valley, excluding farms that had cattle for fattening purposes. Necropsy of the fetus and all related samples were processed in the Laboratory of Parasitology, Faculty of Veterinary Medicine and Animal Science of the Universidad Peruana Cayetano Heredia, Lima, Peru.
PCR
DNA from the fetal brain sample was analyzed using nested PCR on the internal transcribed spacer (ITS1) region of N. caninum. DNA was obtained and quantified by the DNeasy Blood and Tissue Kit (Qiagen, Hilden, Germany) and a NanoDrop Lite spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA), respectively. Nested PCR was carried out using four oligonucleotides, as previously described by Buxton et al. (BUXTON et al., 1998). The Taq PCR Master mix (Qiagen) commercial kit was used. All Qiagen and Thermo Fisher Scientific products were utilized according to the manufacturers’ protocols.
ELISA
Antibody detection against N. caninum was performed for the intrathoracic fluid and serum samples obtained from the aborted fetuses and dairy cattle, respectively. The intrathoracic fluids were centrifuged for 600g for 10 minutes and store in 1.5 ml eppendorf tubes in -80°C. The HerdCheck IDEXX commercial kit was used according to manufacturer’s protocol.
Histology
Fetal tissues (brain, lung, liver, adrenal gland, heart, kidney, thymus, spleen, bone marrow, and intestine) were fixed in 10% neutral buffered formalin, and they were paraffin embedded and stained with hematoxylin and eosin for routine histologic examination. Fetus samples were classified as being positive for N. caninum if they had histopathologic changes compatible (Figure 1) and no compatible with N. caninum infection according Pescador (PESCADOR et al., 2007).
Variables
Outcome variables
We assessed the presence of N. caninum antibodies in dairy cattle. This outcome variable was assessed using the indirect enzyme-linked immunosorbent assay (ELISA) test in 219 bovines. The presence of N. caninum in aborted fetuses were evaluated by PCR, indirect ELISA, and histological analysis. A fetus with at least two positive diagnostic tests (PCR, ELISA, or histology) was considered a positive case.
Independent variables
To evaluate the association between the outcome variable and neosporosis risk factors in dairy cows, we obtained data from the routine health register for cattle. The risk factors evaluated were age and number of births (first birth or multiparous).
Statistical analysis
Statistical analyses were performed in Stata 14.0® (StataCorp, College Station, TX, USA); statistical significance was set at a level of P<0.05. All variables were categorized and presented in absolute and relative frequencies. Aborted fetuses diagnostic were presented with descriptive statistics.The prevalence ratio (PR) and 95% confidence intervals (CI) were estimated to evaluate the association between seroprevalence and risk factors in dairy cattle. Univariate analysis was performed using the chi-squared test.
Results
From the 68 fetuses that were aborted and recollected, 10 (15%) were positive in almost two diagnostic tests and 1 fetus aborted were positive only at least one diagnostic test. PCR and ELISA showed better diagnostic capabilities than histopathologic analysis (Table 1). The brain (5/11) and heart (4/11) showed a high frequency of injuries in histopathologic assay. Also, samples from the fourth and fifth month of gestation showed more cases of neosporosis (Table 2).
Table 2 Number of N. caninum fetuses positive according gestational age and diagnostic technique in dairy cattle from Lima Valley.
| Fetuses (months) | ||||||||||
|---|---|---|---|---|---|---|---|---|---|---|
| 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | Total Positives | ||
| Diagnostic test | ELISA Histology |
- - |
- 1 |
4 2 |
2 1 |
1 1 |
1 - |
1 - |
1 - |
10 5 |
| PCR | 1 | 1 | 4 | 2 | 1 | 1 | - | 1 | 11 | |
| Type of histologic lesion | Compatibles | - | 1 | 2 | 1 | 1 | - | - | - | 5 |
| Non-specific | 1 | - | 1 | - | - | - | - | - | 2 | |
| NLO | - | - | 1 | 1 | - | 1 | - | 1 | 4 | |
NLO: No lesion observed;
PCR: Polymerase chain reaction.
Among 219 dairy bovines included in the study, 46.6% (95% CI: 40.0%-53.3%) were positive for N. caninum antibodies. A significantly higher proportion of N. caninum antibodies were found in older (p<0.001)and multiparous bovines (p=0.017), confirming that age and the number of births were risk factors for this infection (Table 3).
Table 3 Prevalence and factors associated with the presence of antibodies against Neospora caninum in dairy cattle from Lima-Peru.
| Variable | Category | Positive | PR† | CI 95% | P*** | |
|---|---|---|---|---|---|---|
| n** | % | |||||
| Age | 15-24 months | 2 | 10 | Reference | ||
| 2-4 years | 24 | 26.1 | 2.67 | 0.67-5.18 | 0.168 | |
| >4 years | 76 | 71.0 | 7.10* | 4.89-10.67 | <0.001 | |
| N° Births | First birth | 14 | 29.2 | Reference | ||
| Multiparous | 88 | 51.5 | 1.76* | 1.11-2.80 | 0.017 | |
| Antibodies N. caninum | Negative | 117 | 53.4 | - | 46.7-59.9 | - |
| Positive | 102 | 46.6 | - | 40.0-53.3 | ||
†Prevalence ratio;
*p < 0.05, statistical differences with reference group;
**Sample elements
***Chi square p value.
Discussion
The present study aimed to identify the presence of N. caninum in aborted fetuses from dairy cattle. Our findings demonstrate that 15% (10/68) of aborted fetuses were positive in almost two diagnostic assays, at least (Table 1). In Peru, N. caninum cyst records in aborted bovine fetuses showed a moderate proportion of positive cases (55.2%; 16/29), this is higher when compared with the findings from our study (15%). Spontaneous abortions in cattle are strongly associated with N. caninum high seroprevalence herd; and it is associated to dog presence, overcrowding, waste management, high replacement rate (>50%) and low biosecurity in insemination process (HADDAD et al., 2005; HALL et al., 2005; MOEN et al., 1998).
No other infectious abortigenic agents were investigated in this work. Multiple pathogens may produce spontaneous abortions in bovines, such as Leptospira spp., Brucella abortus, bovine viral diarrhea virus, and Aspergillus spp. (RIVERA, 2001). However, this work confirms that neosporosis is present in Peru and is associated with abortions in dairy cows in the Lima Valley.
Seroprevalence of N. caninum in dairy cattle has been reported in different regions of Peru and described their association with aborts.. In the highlands, a seroprevalence of 46.7% was reported (GRANADOS et al., 2014), while in the Amazon rainforest it was 18.8% (PORTOCARRERO et al., 2015), in Lima Valley (Peru central coast) it was 29.6%-69.6% (GAMARRA et al., 2009; SILVA et al., 2002), and in our study it was 46.6%. Rivera reported 55% of aborted fetus with N. caninum presence in differents farms from Lima valley with different diagnostic test (RIVERA, 2001) and our study found 15% of aborted fetus. This prevalence difference would be related to technology level and her size in different region from Peru (BARLING et al., 2000; SENASA, 2011). However, the high prevalence and constant report of aborted fetus in cattle from Peru related to N. caninum presence confirm that seropositive herds present more probability to report aborts than seronegative herds (THURMOND & HIETALA, 1997).
Our study found that cows older than 4 years and multiparous are more likely to possess N. caninum antibodies (Table 3). Other studies also found that age and the numbers of births influence neosporosis (JENSEN et al., 1999; RINALDI et al., 2005). These would be explained by the replacement with no-free N. caninum identification cattle or the use of contaminated reproductive tools (FERRE et al., 2008; SERRANO-MARTÍNEZ et al., 2007). Also in Peru, the lack of diagnosis to N. caninum due to deficient report in semen imported from others cities or countries increment the risk to convert a cow in seropositive by the time or numbers of births (ESCALONA et al., 2010). So, N. caninum identification of multiparous and older cows would be an important control option to neosporosis in stable.
There are different diagnostic techniques for detect N. caninum infection, showing advantages and disadvantages. Nested PCR assay based on the Nc5 and ITS regions demonstrated good performance in different works (GONDIM et al., 2004a; PENA et al., 2007). These genetic regions are the most frequently used to detect N. caninum and are useful for detecting the presence of this protozoo in aborted fetuses autolysis when tissues are frozen. Serology aids in diagnosing fetal neosporosis, since there is no transplacental transfer of antibodies from dam to fetus in cattle. Despite the fact that other reports showed the low sensitivity of fetal serology (GOTTSTEIN et al., 1998; SÖNDGEN et al., 2001), our study found that ELISA performed well when compared with PCR (Table 2). The primary method used to diagnose N. caninum infection in aborted fetuses is histopathology. Fetal lesions such as multifocal, non-suppurative encephalitis and myocarditis are not pathognomonic, but they do allow for a presumptive diagnosis. Moreover, histologic lesions are not exclusive for N. caninum, as they share histologic characteristics with other apicomplexans (DONAHOE et al., 2015; JENKINS et al., 2002; SÁNCHEZ-CASTILLEJA & RODRÍGUEZ-DIEGO, 2018). In conclusion, assays using two or more diagnostic tests may contribute to the successful identification of spontaneous abortion in bovines caused by N. caninum.
In our study, different diagnostic test was applied to detect N. caninum infection, showing PCR and ELISA only differ one sample in diagnostic; meanwhile, histopathology differs in more than 1 (Table 1). Limiting of histopathology is the subjective criterion because need an expert evaluator, in contrast to PCR and ELISA that are objectives diagnostic test (ORTEGA-MORA et al., 2006). ELISA need a high level of antibodies to detect a positive sample; considering immunology system of fetus recollected is in developing, titers of antibodies may not reach cut-off ELISA, need more time to produces more immunoglubuline or the parasite burden was low (ALMERÍA et al., 2016; PEREIRA-BUENO et al., 2003) PCR diagnostic performance is very high, detecting presence of small amounts of N. caninum DNA in large quantity of sample; moreover, PCR detect DNA in tissues of autolysed fetuses (VAN MAANEN et al., 2004). In conclusion, PCR in our study demonstrated be more sensitivity than ELISA in aborted fetus samples.
