Anti-Toxoplasma gondii and anti-Neospora caninum antibodies in dogs with and without neurological signs

ABSTRACT: This study investigated the association between neurological signs as well as plausible risk factors and the seroprevalence of Toxoplasma gondii and Neospora caninum infection in dogs of the Campo Grande region of Mato Grosso do Sul, Brazil. In this study, the dogs were divided into two groups based on the presence and the absence of neurological signs (n=30 in each group). Serological diagnosis was performed using the indirect fluorescent antibody test. In the group with neurological disorders, 23.3% and 30% of the dogs had anti-T. gondii and anti-N. caninum antibodies, respectively. Moreover, three dogs from this group showed co-infection with both protozoa. In the group without neurological signs, 16.7% and 13.3% of the dogs were seropositive for T. gondii and N. caninum, respectively. Although presence of neurological signs was not associated with T. gondii and N. caninum infections (P = 0.747 and P = 0.21, respectively), there was a statistical association between T. gondii seropositivity and peripheral neurological alteration (P = 0.016) among dogs with neurological signs. Raw meat ingestion was the only risk factor associated with the presence of anti-N. caninum antibodies (P = 0.041). Results revealed evidence of exposure to N. caninum and T. gondii in dogs irrespective of the presence of neurological signs. Moreover, this study highlighted the need for serological investigation of T. gondii in dogs with disturbances in peripheral nervous systems and not offering raw meat to animals to avoid the risk of N. caninum infection.

Considering the limited regional data and the importance of T. gondii and N. caninum infection in dog and other species, this study investigated the association between neurological signs (encephalic, spinal, peripheral, or multifocal) as well as plausible infection risk factors of the occurrence of anti-Toxoplasma gondii and anti-Neospora caninum antibodies in the dogs in Campo Grande, Mato Grosso do Sul.
This study included dogs from various regions of Campo Grande, Mato Grosso do Sul, who were treated at the veterinary hospital of the Universidade Federal de Mato Grosso do Sul from April 2019 to December 2019.During this period, 30 dogs presented with neurological signs.After neurological examination, they were categorized according to the anatomical location of the lesion as having in encephalic, spinal, peripheral or multifocal alteration.Additionally, this study included a control group of 30 dogs without neurological signs, who had visited the hospital for problems other than neurological disorders.All dog owners gave their signed consent for participation of the study.
Blood samples were collected for serological diagnosis to verify the presence of anti-N.caninum and anti-T.gondii immunoglobulin G antibodies using an indirect fluorescent antibody test (IFAT), according to previously described methods (CAMARGO, 1974;CONRAD et al., 1993).Only samples that showed fluorescence of the entire tachyzoites surface were considered to have positive IFAT results.Samples with titers ≥ 1:25 and ≥ 1:16 were considered positive for N. caninum and T. gondii infection, respectively.
The owners completed a questionnaire regarding the characteristic of their dogs to identify the possible risk factors associated with T. gondii and N. caninum infections.The questionnaire collected information regarding sex (male/female), age (<5 years/≥5 years), breed (pure breed/mixed breed), place of habitation (urban/rural), presence of other pets (yes/no), contact with wild or wandering animals (yes/no), street access (yes/no), consumption of homemade food (yes/no), ingestion of raw meat (yes/ no), contact with carcasses of other animals (yes/no), pre-existing comorbidities (yes/no), and vaccination (yes, if vaccination was updated/no, if not vaccinated or vaccination was delayed).Other diseases involving neurological changes were not excluded.
The association between the serological test results and aforementioned variables from the epidemiological questionnaire was determined by calculating the odds ratio with a 95% confidence interval.Fisher's exact test was performed for the statistical evaluation of neurological signs and seropositivity frequency.All analyses were performed using the Bioestat 5.0.A P-value < 0.05 was considered statistically significant.
In the group with neurological signs, 23.3% and 30% of the dogs were seropositive for T. gondii and N. caninum, respectively (P = 0.771 for both) (Table 1).In the group without neurological signs, 16.7% and 13.3% of the dogs had antibodies of T. gondii and N. caninum, respectively.The titres ranged from 1:16 to 1:4096 for toxoplasmosis, and from 1:25 to 1:100 for neosporosis.Three dogs from group with neurological disorders (10%) presented co-infection with both the protozoa.
A significant association was observed only between raw meat ingestion and the presence of anti-N.caninum antibodies (P = 0.041).There was no association between other examined variables and the frequency of antibody detection for either of the two protozoa; surprisingly, raw meat ingestion was not associated with T. gondii infection (Table 2).
In our study, the proportion of dogs with neurological signs infected by T. gondii was 23.3%, which was similar to that reported in Paraná (21.08%) (PLUGGE et al., 2011), but higher than that reported in dogs with clinical suspicion of neosporosis in Germany (13%) (KLEIN & MÜLLER, 2001).The proportion of asymptomatic animals infected with T. gondii was 16.7% in our study, which was similar to that reported in other studies conducted in Brazil (12.7% -20.8%) (LANGONI et al., 2013;LOPES et al., 2014); however, this proportion could reach up to 88.5%, as reported by SANTOS et al. (2009).
Presence of antibodies against N. caninum was observed in 13.3% of the dogs without neurological signs, which is similar to what observed in the worldwide canine population (17.14%) (ANVARI et al., 2020), but higher than that in dogs without clinical alterations in Germany (4%) (KLEIN & MÜLLER, 2001).In Brazil, there is a wide variation in the seroprevalence of N. caninum in dogs, ranging from 2.6% in Bahia (SICUPIRA et al., 2012) to 67.6% in Mato Grosso (BENETTI et al., 2009).This could be attributed to several factors, such as sample size, characteristics of the studied population, regional differences, study seasonality, different serological assays, heterogeneous groups, and cut-off titer values (AZEVEDO et al., 2005;ANVARI et al., 2020;GOMES et al., 2020).
There are several reports on animals with neosporosis and toxoplasmosis with neurological signs, such as Horner's syndrome (BOYDELL & BROGAN, 2000), necrotizing cerebellitis (CANTILE   & ARISPICI 2002), encephalomyelitis (PATITUCCI et al., 1997;GERHOLD et al., 2014) and behavioral change (PAPINI, 2009).However, there are only few epidemiological studies on the seroprevalence of N. caninum and T. gondii infections in dogs with neurological signs (PLUGGE et al., 2011;KLEIN & MÜLLER, 2001), since most studies have been conducted on asymptomatic dogs.In this study, the frequency of N. caninum and T. gondii infections was similar among the dogs with neurological signs (30% vs 23.3%; P = 0.771).This finding is different from that of previous studies, which showed that most dogs with neurological clinical signs were seropositive for T. gondii (GIRALDI et al., 2002;PLUGGE et al., 2011;LANGONI et al., 2012).Nevertheless, analyses of the neurological signs by category revealed that peripheral alterations were significantly more often present in T. gondii positive dogs relative to encephalic, spinal or multifocal alterations (P = 0.016) (Table 1).There is an interesting explanation for the peripheral nervous system involvement in toxoplasmosis; namely, there exists a molecular similarity between the pathogenic agent and the proteins or glycolipids in peripheral nerve myelin or the axonal Ranvier's membrane in the initial phase of the infection (HOLT et al., 2011).This allows multiplication of the protozoan and delayed immune response, which, in turn, manifests itself as peripheral neurological alterations (HOLT et al., 2011).Although, we reported statistical significance for the encephalic alteration and T. gondii infection (Table 1), it is noted that the number of seronegative dogs with this clinical sign is much higher (18) in relation to positive ones (2), so that, in our study, encephalic signs were associated with T. gondii seronegative dogs.Ingestion of raw meat was significantly associated with the presence of anti-N.caninum antibodies ( P = 0.041), as previously noted by GAO & WANG (2019) in China.The main route of N. caninum infection in dogs is by the ingestion of food contaminated with tissue cysts.As there was no association between hunting (i.e., contact with wild animals or with carcasses) and antibodies against N. caninum, but there was an association to raw meat ingestion, the infection may probably occur due to the ingestion of beef, as it is the dominant source of meat for dogs in this region of Brazil.
A limitation of this study is its small sample size, due to the low number of neurological cases during the study period.Future studies with larger sample size are warranted for better understanding the epidemiology of the frequency of antibodies against N. caninum and T. gondii in dogs with and without neurological signs in this region.

Table 1 -
Total number, frequency (percentage), and statistical association (Fisher's exact test) of dogs seropositive and seronegative for Toxoplasma gondii and Neospora caninum with and without neurological signs.

Table 2 -
Analysis of risk factors for seropositivity for Toxoplasma gondii and Neospora caninum in 60 dogs from Campo Grande, MS, Brazil.