Serological evaluation of Neospora caninum in pregnant women treated at referral center for prenatal screening in Mato Grosso do Sul, Brazil

Braz J Vet Parasitol 2020; 29(4): e010820 | https://doi.org/10.1590/S1984-29612020097 This is an Open Access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Serological evaluation of Neospora caninum in pregnant women treated at referral center for prenatal screening in Mato Grosso do Sul, Brazil


Introduction
Neospora caninum is an obligate intracellular protozoan that belongs to the phylum Apicomplexa (Dubey et al., 1988). It emerged as a serious disease in cattle and dogs (Dubey, 2003), with canids (Canis domesticus, Canis lupus dingo, Canis latrans, Canis lupus) as definitive hosts (McAllister et al., 1998;King et al., 2010;Gondim et al., 2004;Dubey et al., 2011). The forms of transmission are similar to toxoplasmosis and can be horizontal or vertical. The horizontal form occurs in herbivores via ingestion of water or food contaminated by oocysts and in carnivores via ingestion of tissues infected by tachyzoites or tissue cysts. Vertical transmission occurs transplacentally (Dubey et al., 2007).
Serological and molecular studies of N. caninum in humans and particularly in pregnant women are still scarce in the literature. As there is a report of detection of N. caninum DNA in two human umbilical cord blood samples (Duarte et al., 2020) and this parasite is so similar to T. gondii (Barratt et al., 2010;Hemphill et al., 1999) which is responsible for congenital toxoplasmosis, it is important to investigate the prevalence of N. caninum in pregnant women. For this reason, the objective of this study was to detect anti-N. caninum antibodies in pregnant women treated at a referral center for prenatal screening in the state of Mato Grosso do Sul, Brazil.

Sample acquisition and data collection
The study was approved by the Ethics Committee on Research Involving Human Subjects of the Federal University of Mato Grosso do Sul (Universidade Federal de Mato Grosso do Sul -UFMS) on November 3, 2016, under document n. 1,804,047. A total of 188 serum samples from 46 municipalities of the state of Mato Grosso do Sul were provided by the Instituto de Pesquisa, Ensino e Diagnósticos da APAE de Campo Grande (IPED/ APAE), located in Campo Grande, Mato Grosso do Sul. These samples were divided into three groups: group I contained samples from 99 pregnant women serologically positive for toxoplasmosis in the active phase with IgG/IgM positivity; group II contained samples from 33 human immunodeficiency virus (HIV)-positive pregnant women; and group III contained samples from 56 pregnant women positive for HIV and IgG toxoplasmosis antibodies. Data were collected from the reports provided by IPED/ APAE, which included the results of the prenatal screening tests.

IFA test
Indirect fluorescent antibody (IFA) testing for the detection of anti-N. caninum antibodies was performed using the commercial diagnostic kit Imunoteste® Neospora (RIFI) (Imunodot Diagnostics, Jaboticabal, São Paulo, Brazil) following the manufacturer's recommendations, with modifications. The serum samples were initially diluted 1:50, and positive and negative human serum controls established previously during work conducted at the laboratory were used (Oshiro et al., 2015). Human anti-IgG and anti-IgM fluorescent conjugates (1:100 dilution; conjugated with fluorescein isothiocyanate -Sigma-Aldrich, St. Louis, Missouri, USA) were used for the serological tests. The protocol for IFA test was adapted from Paré et al. (1995).

Western blot analysis
For the western blot, a recombinant partial sequence of the NcSRS2 protein (Nc-p43) was used, which presents approximately 29 KDa and the protocol was adapted from Lima et al. (2007).

Statistical analysis
The collected data were tabulated and analyzed using the statistical software IBM SPSS Statistics, version 20 (IBM Inc., Chicago, Illinois, USA). The χ 2 test were used to evaluate associations between the serological results for N. caninum. P values ≤ 0.05 were considered statistically significant. When the value of the expected frequencies was smaller than five Fisher's exact test was performed.

Results
The analyzed samples came from 46 of the 79 municipalities ( Figure 1) in the state of Mato Grosso do Sul. In group I, the pregnant women from whom the samples were obtained had a mean age of 23.18 ± 6.3057 years, 3/8 Serological evaluation of Neospora caninum and according to the prenatal screening reports, 6% (6/99) of the women were positive for syphilis and 1% (1/99) for hepatitis B. In group II, the mean age was 23.21 years ± 6.1173, and 9.09% (3/33) were positive for syphilis. In group III, the mean age was 26.58 years ± 6.7548 years, and 14.2% (8/56) were positive for syphilis.
There was a significant association between age and N. caninum-positive cases in group III (Table 1). In group I, 30 samples were from the state capital of Campo Grande, with 11 positive for N. caninum (IgG and/or IgM), and the remaining 69 samples were from other cities in the interior of the state, 19 (with two samples positive for both antibodies) of which were positive for N. caninum (IgG and/or IgM). In group II, 17 samples were from the capital Campo Grande, of which 3 were positive for N. caninum (IgG), and the other 16 samples were from the other cities in the interior, 4 of which were positive for N. caninum (IgG). In group III, 21 samples were obtained from the capital Campo Grande, 7 of which were positive for N. caninum (IgG), and the remaining 35 samples were from other cities in the interior, 8 of which were positive for N. caninum (IgG and/or IgM) ( Table 2).

IFA test
Of the 99 analyzed samples from group I, 23 (23.2%) were positive for IgG antibodies, and 9 (9.09%) were positive for anti-N. caninum IgM antibodies as determined by IFA test. In group II, of the 33 analyzed samples, 7 (21.2%) were positive for IgG only. In group III, of the 56 analyzed samples, 13 (23.2%) were positive for IgG antibodies, and 2 (3.5%) were positive for anti-N. caninum IgM antibodies (Tables 2 and 3). According to the Fisher's exact test for the presence of IgG and IgM antibodies, the group II (p = 0.05), and III (p = 0.02) showed a difference in the proportion of positive and negative results at p ≤ 0.05.

Western blot analysis
The seropositivity for N. caninum IgG was confirmed by western blot (Table 3) analysis by testing all sera positive according the IFA test. The tested sera showed reactivity with a 29-kDa protein. Group I showed 82.6% IgG positivity (19/23); group II showed 85.7% IgG positivity (6/7); and group III showed 92.3% IgG positivity (12/13).

Discussion
In the present study, positivity for N. caninum was demonstrated by the IFA test and western blot analysis, which strengthens these findings because the methods are different but complementary serological techniques (Tranas et al., 1999).
For the IFA test, only 23.2% of the samples from group I were positive, indicating the low probability of crossreactivity with T. gondii, whereas 76.8% were negative for N. caninum. In group III, of the 56 samples positive for anti-T. gondii IgG antibodies, 76.7% were negative for N. caninum. These findings corroborate the literature reporting minimal or no cross-reactivity, when dilutions equal to or greater than 1:50 are used in the IFA test (Lobato et al., 2006;Tranas et al., 1999).
In groups II and III, 21.2% and 23.2% of the samples, respectively, were positive for N. caninum IgG antibodies. N. caninum can behave as an opportunistic pathogen in immunocompromised patients (Oshiro et al., 2015;Lobato et al., 2006), and some studies have reported similar rates in HIV-positive patients. In a study by Oshiro et al. (2015) 26.1% of samples from Mato Grosso do Sul and 31.2% for samples from the state of Paraná were positive, whereas in the state of Minas Gerais, Lobato et al. (2006) found that 38% of samples from HIV-positive patients and 6% of samples from healthy individuals were positive, demonstrating that the positivity rate is lower in healthy individuals. The positivity rates may vary according to the technique used and the sample size.
Positivity for anti-N. caninum IgM was observed via IFA test, with 9 samples positive in group I and 2 samples positive in group III. In the literature, there are no data on anti-N. caninum IgM in humans, and the role of the humoral immune response has not yet been elucidated in individuals exposed to the parasite. However, the presence of specific IgM antibodies is indicative of recent infection. A study conducted in the laboratory with mice showed the production of N. caninum-specific IgM on the seventh day after challenge, followed by IgG production starting on the fourteenth day (Teixeira et al., 2005).
There was a significant association between age and N. caninum positive cases in group III, but there are no data in the literature relating positivity to age in humans. However, according to Dubey et al. (2012), one of the main risk factors for T. gondii infection in Brazil is advanced age, as there is increased exposure to the parasite. The significant association between positivity for N. caninum and the proportion of pregnant women positive and negative for only IgG in each group is indicative of past infection.
Reactivity to the N. caninum surface antigen rNcSRS2 (Nc-p43) via western blot, 82.6% (19/23) for group I, 85.7% (6/7) for group II and 92.3% (12/13) for group III, demonstrates that these samples are truly positive because rNcSRS2 (Nc-p43) is highly immunogenic and well-conserved (Howe et al., 1998). Oshiro et al. (2016) used this same protein to develop an indirect serological test (ELISA) to detect anti-N. caninum antibodies in humans and showed sensitivity of 100% and specificity of 90%. In a study carried out by Nam et al. (1998) with human samples of negative and positive sera for T. gondii, the reactivity for a 43 KDa surface protein was observed in the western blot for N. caninum. Three samples showed reactivity, two from the positive group for T. gondii and one from the negative group. This protein seems to be the same used in the present study, however it is not clear in what conditions the western blot was performed and we must take into account that in the present study the recombinant partial sequence of the respective protein was used.
According to Howe & Sibley (1999), the NcSRS2 protein (Nc-p43) has 35 KDa in non-denaturing conditions (without β-mercaptoethanol) and proteins with similar molecular weight have been reported by Lobato et al. (2006) and Tranas et al. (1999) in human sera. Each host species is able to develop its own standard for anti-N. caninum antibodies and variations in the techniques used for the gel, electrophoresis, sample preparation, antigen sources and antibody detection can interfere with the molecular weight variation (Lobato et al., 2006). Studies conducted by Hemphill et al. (1997) and Howe et al. (1998) revealed that the partial nucleotide sequence of the gene encoding NcSRS2 (Nc-p43) has sequence homology with the SRS2 gene from T. gondii, but it was determined by immunotransfer that cross reactivity with T. gondii appears to be insignificant (Bjerkas et al., 1994;Paré et al., 1998;Bae et al., 2000).
Regional conditions must be taken into account so that there is a better understanding of the interaction between antigen and antibody for N. caninum (Lima et al., 2007). Although the rNcSRS2 protein (Nc-p43) did not cross-react with T. gondii in the serum of cattle, dogs and sheep (Borsuk et al., 2011;Pinheiro et al., 2015), further studies are needed to assess the interaction of this antigen with antibodies, from human samples positive for T. gondii.
Neospora caninum is transmitted very efficiently by the transplacental route in cattle (Dubey, 2003), and neosporosis is an important cause of abortion in these animals (Barros et al., 2011). According to data from the Brazilian Institute of Geography and Statistics (IBGE, 2018), Mato Grosso do Sul has the fourth largest cattle herd in Brazil. The seroprevalence for N. caninum in cattle in the state ranged from 14.9% to 43% (Ragozo et al., 2003;Andreotti et al., 2004;Oshiro et al., 2007). A study by Benetti et al. (2009) found a seroprevalence of N. caninum of 10.5% in rural workers and 53.5% in cattle in the state of Mato Grosso. The possibility that N. caninum can infect humans should not be ruled out, as serological evidence in humans is well described in the literature, suggesting exposure to the parasite (Oshiro et al., 2015;Ibrahim et al., 2009;Lobato et al., 2006;Tranas et al., 1999).
An in vitro study showed N. caninum is able to infect human cervical cells and trophoblasts (Carvalho et al., 2010). In experimental studies conducted with nonhuman primates, infection by N. caninum was similar to that induced by T. gondii, indicating susceptibility to transplacental infection and fetal lesions (Ho et al., 1997;Barr et al., 1994).
Although viable N. caninum parasites have not yet been reported in human tissues, DNA was detected for the first time in two umbilical cord blood samples in a study conducted by Duarte et al. (2020) in Brazil. In Spain, among 600 human samples analyzed, none showed positivity for N. caninum (Calero-Bernal et al., 2019). It is not clear whether N. caninum is capable of causing a disease similar to infection by T. gondii and further studies are needed to clarify this possibility.
The serological results of the present study suggest that exposure of these pregnant women to the N. caninum parasite and the presence of IgM antibodies are indicative of recent infection. The role of N. caninum in human pregnancies is not yet clear, but it is known that in mammals such as cattle, sheep and goats, can cause abortion and fetal resorption and mummification; thus, we cannot rule out the possibility that N. caninum is potentially harmful to human pregnancies. Further studies are needed to establish the possibility of active infection.