Seroprevalence of Anti-Neospora caninum and Anti-Toxoplasma gondii Antibodies in Cattle Intended for Human Consumption in the State of Paraíba, Brazil

Simple Summary

This study investigated the infection by two important parasites, Neospora caninum and Toxoplasma gondii, in cattle intended for human consumption in the state of Paraíba, Northeastern Brazil. Blood samples from 110 animals were analyzed using the Indirect Fluorescence Antibody Test. The results showed that 8.2% of the animals were infected with N. caninum and 18.2% with T. gondii. The analysis identified that the extensive farming system is a significant risk factor for both infections. For N. caninum infections, cows showed a higher risk, whereas for T. gondii infection, the frequent animal purchase without quarantine and the lack of separation between young and adult animals were associated with the infections. We conclude that these infections are present in herds and represent a risk to both animal health, by causing reproductive losses, and public health, in the case of T. gondii, which can be transmitted through the consumption of undercooked contaminated meat.

Abstract

We aimed to assess the seroprevalence of anti-Neospora caninum and anti-Toxoplasma gondii antibodies in cattle intended for human consumption in Paraíba, Brazil, and the associated risk factors. A total of 110 serum samples from slaughtered cattle were analyzed using the Indirect Fluorescence Antibody Test (IFAT), with cut-off points of 1:200 for N. caninum and 1:64 for T. gondii. Seroprevalence was 8.2% (9/110) for N. caninum (titers 1:200–1:6400) and 18.2% (20/110) for T. gondii (titers 1:64–1:512). Risk factor analysis revealed that the variable female sex (cows) and the extensive farming system were statistically significantly associated with seroprevalence for N. caninum. Whereas for T. gondii, extensive farming, frequent animal purchase, and the lack of separation between calves and adult cattle were statistically significant. These findings demonstrate the circulation of these parasites in herds, with implications for animal and public health, indicating a potential risk of transmission to definitive hosts and humans through the consumption of raw or undercooked infected meat.

1. Introduction

Cattle are affected by coccidia, obligatory intracellular parasites of the phylum Apicomplexa. Among them, Neospora caninum, the causative agent of neosporosis, stands out as one of the main reproductive diseases affecting these animals [1], as well as Toxoplasma gondii, the causative agent of toxoplasmosis, a cosmopolitan zoonosis of great relevance that can be transmitted to humans through the ingestion of raw or undercooked meat [2].

Neosporosis in cattle is associated with conditions such as fetal mummification, stillbirths, the birth of weak calves, and abortions, and it is considered one of the main causes of infectious abortion in cattle herds worldwide [3]. Studies have reported seropositivity rates for N. caninum in cattle ranging from 9.1% to 91.2% in Brazil [4,5]. Economic losses caused by this disease, considering both dairy and beef herds, are estimated to exceed US$150 million in Brazil [6]. Although there is still no concrete evidence regarding its zoonotic risks to humans, several studies have reported the presence of antibodies against the parasite, and its DNA has already been detected in the umbilical cord of pregnant women [7,8], suggesting possible implications for human health.

Toxoplasmosis is a parasitic infection with high prevalence in both animals and humans [9]. T. gondii has the ability to infect most homeothermic species and form tissue cysts [10]. It is estimated that more than one-third of the human population is infected by this parasite [11]. In Brazil, infection rates of T. gondii in cattle can reach up to 89.1%, depending on the region studied and the diagnostic technique employed, which may be indirect, such as Indirect Fluorescence Antibody Test (IFAT), or Enzyme-Linked Immunosorbent Assay (ELISA), or direct, such as Polymerase Chain Reaction (PCR) [12]. The consumption of raw or undercooked meat is an important vehicle for the transmission of toxoplasmosis to humans, raising concerns from a public health perspective [13,14]. According to a worldwide survey by Dubey [15] on human toxoplasmosis outbreaks (1966–2020), Brazil stood out with the highest number of recorded events. Within the country, seven major outbreaks across different states totaled more than 1350 laboratory-confirmed cases. The clinical presentation of these cases was diverse, ranging from abortions to documented instances of congenital transmission.

Considering the reproductive losses associated with cattle neosporosis and the zoonotic potential of toxoplasmosis, which poses risks to human health through the consumption of contaminated meat, the objective of this study was to determine the seroprevalence of anti-N. caninum and anti-T. gondii antibodies using IFAT in cattle slaughtered for human consumption in the state of Paraíba, as well as to investigate the risk factors associated with these infections.

2. Materials and Methods

Study Area and Sampling

Serological samples were collected during bleeding of cattle from municipal slaughterhouses in the state of Paraíba. To determine the minimum sample size, a simple random sampling calculation was applied [16].

$$n={\displaystyle \frac{{\mathrm{Z}}^2\times \mathrm{P}(1-\mathrm{P})}{d^2}}$$

(1)

where

n = number of sampled animals;

Z = value of the normal distribution for a 95% confidence level;

P = expected prevalence of 18.1% for anti-N. caninum [5] and 18% for anti-T. gondii [17];

d = absolute error of 10%.

The minimum number of samples to be evaluated, as determined by sample size calculation, was 57. However, for convenience, 110 samples were collected. The slaughterhouses used were located in the municipalities of Campina Grande, Itapororoca, Mamanguape, Patos, Santa Rita, and Sapé (Figure 1).

The samples were collected in pre-identified glass tubes without anticoagulant for serum extraction. For transportation, they were placed in a thermal box with ice, maintaining the temperature between 2 and 8 °C until arrival at the laboratory.

Serological analyses were performed using the IFAT, as described by Camargo [18]. For the detection of anti-T. gondii IgG antibodies, tachyzoites of the ME-49 strain fixed on fluorescence microscopy slides were used, adopting a cutoff point of 1:64 as the criterion for positivity [19]. For N. caninum, the protocol established by Gondim et al. [20] was followed, using cell culture-derived tachyzoites of the Nc-1 strain as antigen and a cutoff titer of 1:200 [21]. Complete peripheral fluorescence of the tachyzoites, observed under a fluorescence microscope, was considered the criterion for confirming seropositivity. Positive samples were subjected to titration through twofold serial dilutions until complete absence of fluorescence, at which point they were classified as negative [22].

A questionnaire was completed with the following information: breed (purebred or crossbred), age (≤24 months, >24 months), sex (male or female), contact with cats (yes or no), contact with dogs (yes or no), provision of treated water (yes or no), presence of maternity pens (yes or no), separation of young and adult animals (yes or no), frequent purchase of animals (yes or no), quarantine of newly acquired animals (yes or no), production system (extensive or semi-intensive), history of abortion in the last 12 months (yes or no), and regular veterinary assistance (yes or no).

Descriptive statistical analysis was initially employed to calculate the frequencies of seropositivity obtained through IFAT. To investigate the factors associated with the seroprevalence of anti-N. caninum and anti-T. gondii antibodies, data from the epidemiological questionnaires were subjected to a two-step analytical approach. First, a univariate analysis was performed, in which each independent variable was individually tested against the dependent variable using Fisher’s exact test. Variables with a p-value ≤ 0.05 were selected for the subsequent step. Next, a multivariate analysis was carried out through multiple logistic regression, adopting a significance level of p ≤ 0.05 to identify risk factors independently associated with the infections. All analyses were performed using GraphPad Prism software, version 8.0.1.

3. Results

Among the 110 cattle serum samples analyzed, 8.2% (9/110) tested positive for anti-N. caninum antibodies by IFAT, with titers ranging from 1:200 to 1:6400 (Figure 2). Three animals showed high titers (≥1:800). For anti-T. gondii antibodies, the prevalence was 18.2% (20/110), with titers ranging from 1:64 to 1:512.

The variables obtained from the epidemiological questionnaires are presented in

Table 1. Univariate analysis of factors associated with seropositivity for anti-N. caninum and anti-T. gondii antibodies in cattle intended for human consumption in the state of Paraíba, Brazil.

Variables		N. caninum		T. gondii
	Total	Positive (%)	p	Positive (%)	p
Sex
Male	53	0 (0)	0.003 *	14 (26.4)	0.046 *
Female	57	9 (15.8)		6 (10.5)
Age
≤24 months	38	2 (5.3)	0.7161	4 (10.5)	0.193
>24 months	72	7 (9.7)		16 (22.2)
Breed
Pure	19	1 (5.3)	>0.999	3 (15.8)	>0.999
Crossbred	91	8 (8.8)		17 (18.7)
History of abortion in the last 12 months
Yes	68	6 (8.8)	>0.999	16 (23.5)	0.078
No	42	3 (7.1)		4 (9.5)
Contact with cats
Yes	74	6 (8.3)	>0.999	14 (18.9)	>0.999
No	36	3 (8.1)		6 (16.7)
Contact with dogs
Yes	83	7 (8.4)	>0.999	13 (15.6)	0.256
No	27	2 (7.4)		7 (25.9)
Provision of treated water
Yes	34	4 (11.8)	0.454	5 (14.7)	0.603
No	76	5 (6.6)		15 (19.7)
Frequent purchase of animals
Yes	20	5 (10.9)	0.487	14 (30.4)	0.006 *
No	90	4 (6.2)		6 (9.4)
Separation of young and adult animals
Yes	70	5 (7.1)	0.721	7 (10)	0.005 *
No	40	4 (10)		13 (32.5)
Farming system
Extensive	39	7 (18)	0.009 *	16 (41)	<0.001 *
Semi-intensive	71	2 (2.8)		4 (5.6)
Veterinary assistance
Yes	32	3 (9.4)	0.718	6 (18.7)	>0.999
No	78	6 (7.7)		14 (18)
Presence of maternity pens
Yes	59	2 (3.9)	0.172	5 (9.8)	0.047 *
No	51	7 (11.9)		15 (25.4)

* Variables with a p-value ≤ 0.05 according to the chi-square or Fisher’s exact test.

. A univariate analysis was performed, and those showing statistical significance (p ≤ 0.05) were considered for subsequent multivariate analysis. For N. caninum, the variables included were: sex, production system, and presence of a maternity pen. For T. gondii, the selected variables were: production system, regular acquisition of animals, and separation of young and adult animals.

The multivariate analysis (

Table 2. Multivariate analysis of factors associated with seropositivity for anti-N. caninum and anti-T. gondii antibodies in cattle intended for human consumption in the state of Paraíba.

Variables	Prevalence Ratio	IC 95%	p
	anti-N. caninum antibodies
Sex (Female)	4.8	1.2–19.1	0.025 *
Farming system (Extensive)	5.1	1.5–17.3	0.009 *
	anti-T. gondii antibodies
Farming system (Extensive)	4.3	1.4–13.2	0.011 *
Frequent purchase of animals (Yes)	3.9	1.2–12.8	0.023 *
Separation of young/adult animals (No)	3.1	1.1–8.7	0.032 *

* Variables with a p-value ≤ 0.05.

) identified the following risk factors: female sex and extensive farming system for N. caninum; and extensive farming system, frequent purchase of animals, and lack of separation between young and adult cattle for T. gondii.

4. Discussion

The results of the present study demonstrate significant circulation of N. caninum (8.2%) and T. gondii (18.2%) in cattle slaughter and intended for human consumption in the state of Paraíba, Brazil, corroborating the importance of these parasites for regional livestock and indicating a potential public health risk. The presence of animals with high titers suggests recent infections or reactivations, a scenario in which parasitic replication is more active and the tissue cyst load in meat may be elevated, increasing the zoonotic risk [12,23,24].

The seroprevalence of N. caninum found in this study (8.2%) falls within the wide range reported in Brazil, which can reach up to 90% depending on the region and production system [4]. Similar values were reported by Guerra et al. [25] in Paraná (9.2%), while studies within Paraíba, such as Maia et al. [5], reported a higher prevalence of 17.8%. This disparity can be attributed to the epidemiological particularities of each herd, including the density of definitive hosts and management practices [26]. The fact that our study focused specifically on slaughter animals, generally younger, rather than dairy herds, may partially explain the lower prevalence observed, a factor previously demonstrated in studies comparing seroprevalence across herds with different production purposes [27]. Furthermore, parasite circulation is corroborated by molecular evidence, such as the detection of N. caninum DNA by PCR in 14.3% (5/35) of bovine fetal brains from asymptomatic pregnant dams submitted to slaughter [28], demonstrating the occurrence of active infections and the maintenance of the parasite in the bovine population, even in the absence of overt clinical signs.

For T. gondii, the prevalence of 18.2% aligns with recent studies in the Northeast region, where 18% was reported in cows in Paraíba and 11.8% in Bahia [17,29]. However, it is considerably lower than the 89.1% reported by Gomes et al. [12]. This wide variability reinforces that the epidemiology of cattle toxoplasmosis is complex and hyperendemic in specific microregions, influenced by factors such as the presence of felines, management practices [30], and, critically, climatic conditions that affect oocyst sporulation and survival. In a molecular study analyzing 100 brains from beef cattle, T. gondii DNA was identified in 5% of the samples [31], providing direct evidence of the parasite’s circulation.

Silva et al. [32] demonstrated that conditions in the semi-arid Northeast, characterized by high temperatures and low relative humidity, are decisive for the rapid inactivation of oocysts, which can be destroyed within 36 h. This environment creates a natural barrier to environmental transmission. In contrast, the South and Southeast regions of Brazil, with temperate or more humid tropical climates and soils with greater water retention capacity, offer ideal conditions for prolonged oocyst survival [15]. Therefore, extreme climatic conditions represent a key environmental factor explaining, in part, the lower seroprevalences generally found in herds from hot and dry regions, as observed in our study, compared to the high rates reported in cooler and more humid regions.

Multivariate analysis identified critical risk factors for both infections. The extensive farming system emerged as a significant risk factor for N. caninum and T. gondii. This finding is consistent with the literature, as this system favors cattle contact with pastures and water sources potentially contaminated with oocysts excreted by dogs and felids [33,34]. The difficulty in implementing biosecurity measures and controlling synanthropic fauna in these systems amplifies exposure [35].

Specifically for N. caninum, female sex was an important risk factor, likely due to the longer lifespan of females in herds, whether for replacement, lactation, or rearing, extending their window of exposure to environmental oocysts compared to males, which are typically sent for early slaughter [36]. Additionally, vertical transmission is a primary route for maintaining N. caninum in herds, creating a cycle where infected females perpetuate the parasite in their offspring, which in turn remain in the herd [27]. However, our data did not find a significant association with abortion history, possibly due to sample size or because many abortions caused by neosporosis may go unreported.

For T. gondii, two management factors were particularly relevant: frequent purchase of animals and the lack of separation between young and adult cattle. Introducing animals without prior quarantine or testing is a classic pathway for the entry of infectious diseases into a closed herd [37]. The lack of separation between calves and adults can facilitate parasite transmission. First, studies demonstrate that raw milk can be a significant route of T. gondii transmission, especially during the acute phase of infection, and co-mingling allows calves to consume this milk [38,39]. Second, as it is common for adult animals to receive concentrate-based supplementation, feed residues can attract rodents. These, in turn, attract felids, which are the definitive hosts of the parasite and can contaminate the environment with oocysts [29]. T. gondii sporulated oocysts are extremely resistant in favorable environments, surviving for months in soil and water, thereby turning common grazing or living areas into permanent foci of infection [31].

The public health implications are notable, particularly for T. gondii. The detection of antibodies indicates that the tissues of these animals may harbor parasite cysts. Consumption of raw or undercooked beef from these animals represents a transmission route to humans [14,15]. Brazil, as highlighted by [16], experiences numerous foodborne toxoplasmosis outbreaks, making herd surveillance crucial. Although N. caninum transmission via meat is not confirmed in humans, the presence of the parasite in cattle tissues poses a risk to dogs, its definitive hosts, which, upon infection, complete the cycle and maintain the parasite in the environment [26].

5. Conclusions

There was demonstrated the significant presence of anti-N. caninum and anti-T. gondii antibodies in cattle slaughtered for human consumption in the state of Paraíba, Brazil, indicating exposure of the animals to these protozoa and highlighting the importance of the infections in local livestock. Therefore, measures such as controlling the presence of definitive hosts in the environment, restricting their contact with herds, adopting proper sanitary management practices, and implementing serological monitoring are essential to minimize the impact of these infections on animal and public health, thereby promoting food safety in livestock production.