Evaluation of abortions spontaneously induced by Neospora caninum and risk factors in dairy cattle from Lima, Peru

Avaliação de abortos induzidos espontaneamente por Neospora caninum e fatores de risco em bovinos leiteiros de Lima, Peru

Marcos Enrique Serrano-Martínez Cesar Abel Burga Cisterna Roberto Carlos Evaristo Romero Marco Antonio Quispe Huacho Alessandra Matienzo Bermabé Luis Antonio Llanco Albornoz About the authors

Abstract

Our objective was to identify the direct and indirect presence of Neospora caninum in dairy cattle and their aborted fetuses from Lima, Peru. A total 219 blood samples obtained from dairy cattle with records of spontaneous abortion were collected to detect antibodies against N. caninum in serum with indirect ELISA and search for risk-factor associations. 68 fetal aborted tissue samples of these cows were analyzed by PCR, indirect ELISA and histopathology assay to detect N. caninum presence. The prevalence ratio (PR)

and 95% confidence intervals (CI) were estimated. Univariate analysis was performed using the chi-squared test. Among the 68 aborted fetuses collected, 10 (15%) were positive in at least two diagnostic tests. Among 219 serum samples, 46.6% (95% CI: 40.0%-53.3%) were positive. Cows with 4 years or older (PR: 7.10; 95% CI: 4.89-10.67) and multiparous (PR: 1.76; 95% CI: 1.11-2.80) were found to be more likely to possess N. caninum antibodies. This study detects presence of N. caninum in dairy cattle and their aborted fetus from Lima valley, suggesting biosecurity management improve to neosporosis control.

Keywords:
Aborts; neosporosis; bovine; diagnostic; Peru

Resumo

O objetivo do trabalho foi diagnosticar a presença direta e indireta de Neospora caninum em fetos abortados e de soros de bovinos leiteiros de Lima, Peru. Um total de 219 amostras de sangue obtido de vacas leiteiras, com registros de abortos espontâneos, foi coletado para detectar anticorpos contra N. caninum no soro (pelo ELISA indireto), e para estudar associações com fatores de risco. Foram examinadas 68 amostras de tecido fetal abortado das vacas soropositivas pela PCR, ELISA indireto e histopatologia para determinar a presença de N. caninum. A taxa de prevalência (PR) e o intervalo de confiança (CI) de 95% foram estimados. Análise univariada foi realizada usando o teste de Qui-quadrado. Entre os 68 fetos abortados, 10 (15%) foram positivos em pelo menos dois testes diagnósticos. Entre as 219 amostras de soro, 46,6% (95% IC: 40,0%-53,3%) foram positivas. Vacas com 4 anos ou mais de idade (RP: 7,10; 95% IC: 4,89-10,67) e multíparas (RP: 1,76; 95% IC: 1,11-2,80) apresentaram maior frequência de anticorpos contra N. caninum. Este estudo demonstrou a presença de N. caninum em bovinos leiteiros e em fetos abortados oriundos do vale de Lima, sugerindo a implementação de medidas de biossegurança, para o controle da neosporose.

Palavras-chave:
Aborto; neosporosis; bovino; diagnóstico; Peru

Introduction

Neospora caninum is an Apicomplexa identified as a causal agent of reproductive problems in cattle worldwide (DUBEY, 2003Dubey JP. Review of Neospora caninum and neosporosis in animals. Korean J Parasitol 2003; 41(1): 1-16. http://dx.doi.org/10.3347/kjp.2003.41.1.1. PMid:12666725.
http://dx.doi.org/10.3347/kjp.2003.41.1....
). In their life cycle, dogs (MCALLISTER et al., 1998McAllister MM, Dubey J, Lindsay DS, Jolley WR, Wills RA, McGuire AM. Dogs are definitive hosts of Neospora caninum. Int J Parasitol 1998; 28(9): 1473-1478. http://dx.doi.org/10.1016/S0020-7519(98)00138-6. PMid:9770635.
http://dx.doi.org/10.1016/S0020-7519(98)...
), coyotes (GONDIM et al., 2004bGondim LFP, McAllister MM, Pitt WC, Zemlicka DE. Coyotes (Canis latrans) are definitive hosts of Neospora caninum. Int J Parasitol 2004b; 34(2): 159-161. http://dx.doi.org/10.1016/j.ijpara.2004.01.001. PMid:15037103.
http://dx.doi.org/10.1016/j.ijpara.2004....
), and grey wolves (DUBEY et al., 2011Dubey JP, Jenkins MC, Rajendran C, Miska K, Ferreira LR, Martins J, et al. Gray wolf (Canis lupus) is a natural definitive host for Neospora caninum. Vet Parasitol 2011; 181(2-4): 382-387. http://dx.doi.org/10.1016/j.vetpar.2011.05.018. PMid:21640485.
http://dx.doi.org/10.1016/j.vetpar.2011....
) are definitive hosts. N. caninum easily survives in many mammals, identifying multiple intermediate hosts such as cows, sheep, goats, deer (CABRAL et al., 2009Cabral AD, Camargo CN, Galleti NTC, Okuda LH, Pituco EM, Del Fava C. Diagnosis of Neospora caninum in bovine fetuses by histology, immunohistochemistry, and nested-PCR. Rev Bras Parasitol Vet 2009; 18(4): 14-19. http://dx.doi.org/10.4322/rbpv.01804003. PMid:20040203.
http://dx.doi.org/10.4322/rbpv.01804003...
; PANADERO et al., 2010Panadero R, Painceira A, López C, Vázquez L, Paz A, Díaz P, et al. Seroprevalence of Toxoplasma gondii and Neospora caninum in wild and domestic ruminants sharing pastures in Galicia (Northwest Spain). Res Vet Sci 2010; 88(1): 111-115. http://dx.doi.org/10.1016/j.rvsc.2009.05.010. PMid:19482324.
http://dx.doi.org/10.1016/j.rvsc.2009.05...
; SALABERRY et al. 2010Salaberry SR, Okuda LH, Nassar AF, Castro JR, Lima-Ribeiro AMC. Prevalence of Neospora caninum antibodies in sheep flocks of Uberlandia county, MG. Rev Bras Parasitol Vet 2010; 19(3): 148-151. http://dx.doi.org/10.1590/S1984-29612010000300004. PMid:20943017.
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; VARASCHIN et al., 2012Varaschin MS, Hirsch C, Wouters F, Nakagaki KY, Guimarães AM, Santos DS, et al. Congenital neosporosis in goats from the State of Minas Gerais, Brazil. Korean J Parasitol 2012; 50(1): 63-67. http://dx.doi.org/10.3347/kjp.2012.50.1.63. PMid:22451736.
http://dx.doi.org/10.3347/kjp.2012.50.1....
), and alpacas (CHÁVEZ-VELÁSQUEZ et al., 2004Chávez-Velásquez A, Álvarez-García G, Collantes-Fernández E, Casas-Astos E, Rosadio-Alcántara R, Serrano-Martínez E, et al. First Report of Neospora caninum Infection in Adult Alpacas (Vicugna pacos) and Llamas (Lama glama). J Parasitol 2004; 90(4): 864-866. http://dx.doi.org/10.1645/GE-260R. PMid:15357084.
http://dx.doi.org/10.1645/GE-260R...
).

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., 2005Hall CA, Reichel MP, Ellis JT. Neospora abortions in dairy cattle: diagnosis, mode of transmission and control. Vet Parasitol 2005; 128(3-4): 231-241. http://dx.doi.org/10.1016/j.vetpar.2004.12.012. PMid:15740860.
http://dx.doi.org/10.1016/j.vetpar.2004....
; MOEN et al., 1998Moen AR, Wouda W, Mul MF, Graat EAM, Van Werven T. Increased risk of abortion following Neospora caninum abortion outbreaks: a retrospective and prospective cohort study in four dairy herds. Theriogenology 1998; 49(7): 1301-1309. http://dx.doi.org/10.1016/S0093-691X(98)00077-6. PMid:10732067.
http://dx.doi.org/10.1016/S0093-691X(98)...
). 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, 1997Thurmond MC, Hietala SK. Effect of congenitally acquired Neospora caninum infection on risk of abortion and subsequent abortions in dairy cattle. Am J Vet Res 1997; 58(12): 1381-1385. PMid:9401685.). Therefore, seropositivity in herds might increase with age or gestation number, and horizontal transmission can influence this event (JENSEN et al., 1999Jensen AM, Björkman C, Kjeldsen AM, Wedderkopp A, Willadsen C, Uggla A, et al. Associations of Neospora caninum seropositivity with gestation number and pregnancy outcome in Danish dairy herds. Prev Vet Med 1999; 40(3-4): 151-163. http://dx.doi.org/10.1016/S0167-5877(99)00048-3. PMid:10423771.
http://dx.doi.org/10.1016/S0167-5877(99)...
; RINALDI et al., 2005Rinaldi L, Fusco G, Musella V, Veneziano V, Guarino A, Taddei R, et al. Neospora caninum in pastured cattle: determination of climatic, environmental, farm management and individual animal risk factors using remote sensing and geographical information systems. Vet Parasitol 2005; 128(3-4): 219-230. http://dx.doi.org/10.1016/j.vetpar.2004.12.011. PMid:15740859.
http://dx.doi.org/10.1016/j.vetpar.2004....
). 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, 2001Gamarra G. Situación actual y perspectivas de la ganadería lechera en la cuenca de Lima. Rev Investig Vet Peru 2001; 12(2): 1-13.; RIVERA, 2001Rivera H. Causas frecuentes de aborto bovino. Rev Investig Vet Peru 2001; 12(2): 117-122.). Several studies have found N. caninum high prevalence in dairy cattle from Lima Valley (GAMARRA et al., 2009Gamarra S, Salazar I, Zarate D, Vargas J. Seroprevalencia de Neospora caninum en una población ganadera lechera de la Cuenca de Lima. Anales Científ UNALM 2009; 70(1): 81-85. http://dx.doi.org/10.21704/ac.v70i1.76.
http://dx.doi.org/10.21704/ac.v70i1.76...
; SILVA et al., 2002Silva P, Chávez A, Rivera H, Casas E. Seroprevalencia de Neospora caninum en Bovinos lecheros del valle de Lima. Rev Investig Vet Peru 2002; 13(2): 51-55. http://dx.doi.org/10.15381/rivep.v13i2.7330.
http://dx.doi.org/10.15381/rivep.v13i2.7...
). Considering N. caninum presence such a risk in reproductive cattle health (DUBEY et al., 2007Dubey J, Schares G, Ortega-Mora L. Epidemiology and control of neosporosis and Neospora caninum. Clin Microbiol Rev 2007; 20(2): 323-367. http://dx.doi.org/10.1128/CMR.00031-06. PMid:17428888.
http://dx.doi.org/10.1128/CMR.00031-06...
), 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., 2006Ortega-Mora L, Fernández-García A, Gómez-Bautista M. Diagnosis of bovine neosporosis: recent advances and perspectives. Acta Parasitol 2006; 51(1): 1-14. http://dx.doi.org/10.2478/s11686-006-0001-0.
http://dx.doi.org/10.2478/s11686-006-000...
) or their ability to be influenced by pregnancy (NOGAREDA et al., 2007Nogareda C, López-Gatius F, Santolaria P, García-Ispierto I, Bech-Sàbat G, Pabón M, et al. Dynamics of anti-Neospora caninum antibodies during gestation in chronically infected dairy cows. Vet Parasitol 2007; 148(3-4): 193-199. http://dx.doi.org/10.1016/j.vetpar.2007.06.032. PMid:17643822.
http://dx.doi.org/10.1016/j.vetpar.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., 2010Al-Qassab SE, Reichel MP, Ellis JT. On the biological and genetic diversity in Neospora caninum. Diversity (Basel) 2010; 2(3): 411-438. http://dx.doi.org/10.3390/d2030411.
http://dx.doi.org/10.3390/d2030411...
; ELLIS, 1998Ellis JT. Polymerase chain reaction approaches for the detection of Neospora caninum and Toxoplasma gondii. Int J Parasitol 1998; 28(7): 1053-1060. http://dx.doi.org/10.1016/S0020-7519(98)00096-4. PMid:9724876.
http://dx.doi.org/10.1016/S0020-7519(98)...
). Histopathology has a low sensitivity for detection when compared to other diagnostic tests; however, it still holds important diagnostic value (DUBEY & SCHARES, 2006Dubey J, Schares G. Diagnosis of bovine neosporosis. Vet Parasitol 2006; 140(1-2): 1-34. http://dx.doi.org/10.1016/j.vetpar.2006.03.035. PMid:16730126.
http://dx.doi.org/10.1016/j.vetpar.2006....
; SÁNCHEZ-CASTILLEJA & RODRÍGUEZ-DIEGO, 2018Sánchez-Castilleja YM, Rodríguez-Diego JG. Identificación de lesiones histológicas coincidentes con Neospora caninum en tejido cerebral de fetos bovinos. Rev Salud Anim 2018; 40(1): 1-4.). 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.

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., 1998Buxton D, Maley SW, Wright S, Thomson KM, Rae AG, Innes EA. The pathogenesis of experimental neosporosis in pregnant sheep. J Comp Pathol 1998; 118(4): 267-279. http://dx.doi.org/10.1016/S0021-9975(07)80003-X. PMid:9651804.
http://dx.doi.org/10.1016/S0021-9975(07)...
). 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., 2007Pescador CA, Corbellini LG, Oliveira EC, Raymundo DL, Driemeier D. Histopathological and immunohistochemical aspects of Neospora caninum diagnosis in bovine aborted fetuses. Vet Parasitol 2007; 150(1-2): 159-163. http://dx.doi.org/10.1016/j.vetpar.2007.08.028. PMid:17904290.
http://dx.doi.org/10.1016/j.vetpar.2007....
).

Figure 1
Fetal brain of an aborted bovine fetus with mild diffuse encephalitis. Diffuse gliosis and focal necrosis surrounded by mononuclear inflammatory cells (H&E-stained sections, magnification is 100x).

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.

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.

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., 2005Haddad JP, Dohoo IR, VanLeewen J. A review of Neospora caninum in dairy and beef cattle--a Canadian perspective. Can Vet J 2005; 46(3): 230-243. PMID: 15884645.; HALL et al., 2005Hall CA, Reichel MP, Ellis JT. Neospora abortions in dairy cattle: diagnosis, mode of transmission and control. Vet Parasitol 2005; 128(3-4): 231-241. http://dx.doi.org/10.1016/j.vetpar.2004.12.012. PMid:15740860.
http://dx.doi.org/10.1016/j.vetpar.2004....
; MOEN et al., 1998Moen AR, Wouda W, Mul MF, Graat EAM, Van Werven T. Increased risk of abortion following Neospora caninum abortion outbreaks: a retrospective and prospective cohort study in four dairy herds. Theriogenology 1998; 49(7): 1301-1309. http://dx.doi.org/10.1016/S0093-691X(98)00077-6. PMid:10732067.
http://dx.doi.org/10.1016/S0093-691X(98)...
).

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, 2001Rivera H. Causas frecuentes de aborto bovino. Rev Investig Vet Peru 2001; 12(2): 117-122.). 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., 2014Granados SZ, Rivera HG, Casas EA, Suárez FA, Arana CD, Chávez AV. Seroprevalencia de Neospora caninum en bovinos lecheros de cuatro distritos del Valle del Mantaro, Junín. Rev Investig Vet Peru 2014; 25(1): 58-64. http://dx.doi.org/10.15381/rivep.v25i1.8468.
http://dx.doi.org/10.15381/rivep.v25i1.8...
), while in the Amazon rainforest it was 18.8% (PORTOCARRERO et al., 2015Portocarrero CM, Pinedo RV, Falcón NP, Chávez AV. Factores de riesgo asociados a la seroprevalencia de Neospora caninum en bovinos naturalmente infectados en la ceja de selva de Oxapampa, Perú. Rev Investig Vet Peru 2015; 26(1): 119-126. http://dx.doi.org/10.15381/rivep.v26i1.10916.
http://dx.doi.org/10.15381/rivep.v26i1.1...
), in Lima Valley (Peru central coast) it was 29.6%-69.6% (GAMARRA et al., 2009Gamarra S, Salazar I, Zarate D, Vargas J. Seroprevalencia de Neospora caninum en una población ganadera lechera de la Cuenca de Lima. Anales Científ UNALM 2009; 70(1): 81-85. http://dx.doi.org/10.21704/ac.v70i1.76.
http://dx.doi.org/10.21704/ac.v70i1.76...
; SILVA et al., 2002Silva P, Chávez A, Rivera H, Casas E. Seroprevalencia de Neospora caninum en Bovinos lecheros del valle de Lima. Rev Investig Vet Peru 2002; 13(2): 51-55. http://dx.doi.org/10.15381/rivep.v13i2.7330.
http://dx.doi.org/10.15381/rivep.v13i2.7...
), 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, 2001Rivera H. Causas frecuentes de aborto bovino. Rev Investig Vet Peru 2001; 12(2): 117-122.) 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., 2000Barling KS, Sherman M, Peterson MJ, Thompson JA, McNeill JW, Craig TM, et al. Spatial associations among density of cattle, abundance of wild canids, and seroprevalence to Neospora caninum in a population of beef calves. J Am Vet Med Assoc 2000; 217(9): 1361-1365. http://dx.doi.org/10.2460/javma.2000.217.1361. PMid:11061391.
http://dx.doi.org/10.2460/javma.2000.217...
; SENASA, 2011Servicio Nacional de Sanidad y Calidad Agroalimentaria – SENASA. Caracterización de la diarrea viral bovina, neosporosis bovina y rinotraqueitis infecciosa bovina en el Peru [online] 2011. [cited 2019 Mar 13]. Buenos Aires: Servicio Nacional de Salud Agraria del Peru. Available from: http://repositorio.senasa.gob.pe/bitstream/SENASA/135/1/2011_SENASA_DVB-neosporosis-IBR.pdf
http://repositorio.senasa.gob.pe/bitstre...
). 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, 1997Thurmond MC, Hietala SK. Effect of congenitally acquired Neospora caninum infection on risk of abortion and subsequent abortions in dairy cattle. Am J Vet Res 1997; 58(12): 1381-1385. PMid:9401685.).

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., 1999Jensen AM, Björkman C, Kjeldsen AM, Wedderkopp A, Willadsen C, Uggla A, et al. Associations of Neospora caninum seropositivity with gestation number and pregnancy outcome in Danish dairy herds. Prev Vet Med 1999; 40(3-4): 151-163. http://dx.doi.org/10.1016/S0167-5877(99)00048-3. PMid:10423771.
http://dx.doi.org/10.1016/S0167-5877(99)...
; RINALDI et al., 2005Rinaldi L, Fusco G, Musella V, Veneziano V, Guarino A, Taddei R, et al. Neospora caninum in pastured cattle: determination of climatic, environmental, farm management and individual animal risk factors using remote sensing and geographical information systems. Vet Parasitol 2005; 128(3-4): 219-230. http://dx.doi.org/10.1016/j.vetpar.2004.12.011. PMid:15740859.
http://dx.doi.org/10.1016/j.vetpar.2004....
). These would be explained by the replacement with no-free N. caninum identification cattle or the use of contaminated reproductive tools (FERRE et al., 2008Ferre I, Serrano-Martínez E, Martínez A, Osoro K, Mateos-Sanz A, del-Pozo I, et al. Effects of re-infection with Neospora caninum in bulls on parasite detection in semen and blood and immunological responses. Theriogenology 2008; 69(7): 905-911. http://dx.doi.org/10.1016/j.theriogenology.2007.11.006. PMid:18336895.
http://dx.doi.org/10.1016/j.theriogenolo...
; SERRANO-MARTÍNEZ et al., 2007Serrano-Martínez E, Ferre I, Martínez A, Osoro K, Mateos-Sanz A, del-Pozo I, et al. Experimental neosporosis in bulls: Parasite detection in semen and blood and specific antibody and interferon-gamma responses. Theriogenology 2007; 67(6): 1175-1184. http://dx.doi.org/10.1016/j.theriogenology.2007.01.010. PMid:17316779.
http://dx.doi.org/10.1016/j.theriogenolo...
). 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., 2010Escalona J, García F, Mosquera O, Vargas F, Corro A. Factores de riesgo asociados a la prevalencia de Neosporosis Bovina en el municipio Bolívar del estado Yaracuy, Venezuela. Zootec Trop 2010; 28(2): 201-211.). 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., 2004aGondim LFP, Laski P, Gao L, McAllister MM. Variation of the internal transcribed spacer 1 sequence within individual strains and among different strains of Neospora caninum. J Parasitol 2004a; 90(1): 119-122. http://dx.doi.org/10.1645/GE-134R. PMid:15040677.
http://dx.doi.org/10.1645/GE-134R...
; PENA et al., 2007Pena HFJ, Soares RM, Ragozo AMA, Monteiro RM, Yai LEO, Nishi SM, et al. Isolation and molecular detection of Neospora caninum from naturally infected sheep from Brazil. Vet Parasitol 2007; 147(1-2): 61-66. http://dx.doi.org/10.1016/j.vetpar.2007.03.002. PMid:17451882.
http://dx.doi.org/10.1016/j.vetpar.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., 1998Gottstein B, Hentrich B, Wyss R, Thür B, Busato A, Stärk KDC, et al. Molecular and immunodiagnostic investigations on bovine neosporosis in Switzerland. Int J Parasitol 1998; 28(4): 679-691. http://dx.doi.org/10.1016/S0020-7519(98)00006-X. PMid:9602392.
http://dx.doi.org/10.1016/S0020-7519(98)...
; SÖNDGEN et al., 2001Söndgen P, Peters M, Bärwald A, Wurm R, Holling F, Conraths FJ, et al. Bovine neosporosis: immunoblot improves foetal serology. Vet Parasitol 2001; 102(4): 279-290. http://dx.doi.org/10.1016/S0304-4017(01)00543-X. PMid:11731071.
http://dx.doi.org/10.1016/S0304-4017(01)...
), 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., 2015Donahoe SL, Lindsay SA, Krockenberger M, Phalen D, Šlapeta J. A review of neosporosis and pathologic findings of Neospora caninum infection in wildlife. Int J Parasitol Parasites Wildl 2015; 4(2): 216-238. http://dx.doi.org/10.1016/j.ijppaw.2015.04.002. PMid:25973393.
http://dx.doi.org/10.1016/j.ijppaw.2015....
; JENKINS et al., 2002Jenkins M, Baszler T, Björkman C, Schares G, Williams D. Diagnosis and seroepidemiology of Neospora caninum-associated bovine abortion. Int J Parasitol 2002; 32(5): 631-636. http://dx.doi.org/10.1016/S0020-7519(01)00363-0. PMid:11943234.
http://dx.doi.org/10.1016/S0020-7519(01)...
; SÁNCHEZ-CASTILLEJA & RODRÍGUEZ-DIEGO, 2018Sánchez-Castilleja YM, Rodríguez-Diego JG. Identificación de lesiones histológicas coincidentes con Neospora caninum en tejido cerebral de fetos bovinos. Rev Salud Anim 2018; 40(1): 1-4.). 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., 2006Ortega-Mora L, Fernández-García A, Gómez-Bautista M. Diagnosis of bovine neosporosis: recent advances and perspectives. Acta Parasitol 2006; 51(1): 1-14. http://dx.doi.org/10.2478/s11686-006-0001-0.
http://dx.doi.org/10.2478/s11686-006-000...
). 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., 2016Almería S, Serrano-Perez B, Darwich L, Domingo M, Mur-Novales R, Regidor-Cerrillo J, et al. Foetal death in naive heifers inoculated with Neospora caninum isolate Nc-Spain7 at 110 days of pregnancy. Exp Parasitol 2016; 168: 62-69. http://dx.doi.org/10.1016/j.exppara.2016.06.009. PMid:27388371.
http://dx.doi.org/10.1016/j.exppara.2016...
; PEREIRA-BUENO et al., 2003Pereira-Bueno J, Quintanilla-Gozalo A, Pérez-Pérez V, Espi-Felgueroso A, Alvarez-García G, Collantes-Fernández E, et al. Evaluation by different diagnostic techniques of bovine abortion associated with Neospora caninum in Spain. Vet Parasitol 2003; 111(2-3): 143-152. http://dx.doi.org/10.1016/S0304-4017(02)00361-8. PMid:12531290.
http://dx.doi.org/10.1016/S0304-4017(02)...
) 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., 2004van Maanen C, Wouda W, Schares G, von Blumröder D, Conraths FJ, Norton R, et al. An interlaboratory comparison of immunohistochemistry and PCR methods for detection of Neospora caninum in bovine foetal tissues. Vet Parasitol 2004; 126(4): 351-364. http://dx.doi.org/10.1016/j.vetpar.2004.08.016. PMid:15567040.
http://dx.doi.org/10.1016/j.vetpar.2004....
). In conclusion, PCR in our study demonstrated be more sensitivity than ELISA in aborted fetus samples.

Conclusion

Neospora caninum was detected in aborted fetus from Lima Valley and seropositivity of cattle with abort records is significantly associated with age and number of births, suggesting this variables be a risk factors to infection with this parasite.

Acknowledgements

This study was supported by Grant: Fondo Innovación, Ciencia y Tecnología (FINCyT-Innóvate Peru), Proyect “Obtención y caracterización del primer aislado de Neospora caninum causante de abortos y mortalidad neonatal en la producción bovina lechera del Peru, con fines inmunodiagnóstico y vacunal” (contrato 167-PNICP-PIAP-2015 FINCYT); Programa Iberoamericano de Ciencia y Tecnología para el Desarrollo (CYTED), support for Red Temática 113RT0469 PROTOZOOVAC: “DIAGNÓSTICO Y CONTROL DE LAS PROTOZOOSIS REPRODUCTIVAS DEL GANADO BOVINO” that facilitated technical capacitation of our researchers. We thank the breeders and thesis students, who collaborated during the collection of samples.

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Publication Dates

  • Publication in this collection
    13 June 2019
  • Date of issue
    Apr-Jun 2019

History

  • Received
    07 Nov 2018
  • Accepted
    13 Mar 2019
Colégio Brasileiro de Parasitologia Veterinária FCAV/UNESP - Departamento de Patologia Veterinária, Via de acesso Prof. Paulo Donato Castellane s/n, Zona Rural, , 14884-900 Jaboticabal - SP, Brasil, Fone: (16) 3209-7100 RAMAL 7934 - Jaboticabal - SP - Brazil
E-mail: cbpv_rbpv.fcav@unesp.br