Correlation between Anti-Toxoplasma gondii IgG Antibodies in Serum and Colostrum of Naturally Infected Sheep and Passive Immunization in Lambs

Abstract

Toxoplasmosis, caused by Toxoplasma gondii, poses a significant threat to sheep flocks, affecting reproductive performance and meat quality, and leading to economic losses. This study aimed to evaluate the correlation between anti-T. gondii IgG antibodies in the serum and colostrum of naturally infected ewes and to assess passive immunity in newborn lambs. Blood and colostrum samples were collected from 162 ewes and 182 lambs across 20 sheep farms in Paraíba, Brazil. Samples were tested for anti-T. gondii and anti-Neospora caninum IgG using indirect fluorescence antibody tests (IFATs), with titers ≥ 1:64 considered positive. Among the ewes, 45.1% tested positive for anti-T. gondii IgG in serum, with titers ranging from 1:64 to 1:16,384. The colostrum from 94.6% of the ewes also tested positive, although 74% had higher titers in their serum than in their colostrum. Concordance between serum and colostrum was high, with a kappa coefficient of 0.950. Lamb serum showed a perfect agreement with maternal colostrum (kappa = 0.962), demonstrating effective passive transfer of antibodies. This study confirms that colostrum is a reliable matrix for detecting anti-T. gondii antibodies and assessing passive immunity in lambs. The high concordance between serum, colostrum, and lamb titers suggests that IFATs on colostrum can be a practical tool for monitoring maternal antibody transfer, contributing to the better management of T. gondii infections in sheep flocks.

1. Introduction

Toxoplasmosis is a disease caused by the protozoan Toxoplasma gondii (Apicomplexa: Sarcocystidae). In sheep flocks, it can lead to various issues, including abortions, neurological symptoms, and the contamination of meat intended for human consumption. Humans can become infected by ingesting viable cysts present in the tissues [1,2]. Toxoplasmosis not only affects the health and productivity of sheep flocks but also poses significant economic losses for sheep farmers [3]. The cost of managing outbreaks, treating affected animals, and the potential loss of the marketability of meat and leather products can be substantial [4]. Moreover, consumer confidence can be severely impacted if the presence of T. gondii in sheep products is not adequately controlled [5].

Brazilian sheep flocks are primarily located in the northeastern and southern regions [6]. Studies conducted in the northeast region of Brazil have shown that approximately 30% of sheep meat intended for human consumption comes from animals that have tested seropositive for T. gondii [2]. Additionally, the parasite can be transmitted vertically, resulting in some lambs being naturally infected from birth [7].

Sheep production primarily focuses on meat and leather, requiring high reproductive rates and ensuring that the final product is free from zoonotic cysts. Toxoplasma gondii negatively impacts both reproductive rates and meat quality, posing a significant threat to production [8].

The transmission of T. gondii in sheep flocks occurs through the ingestion of food or water contaminated with viable oocysts excreted by infected cats [8]. In animals with low immunity, the parasite can reactivate, leading to the recurrence of clinical signs and the formation of new tissue cysts as bradyzoite infections cycle back [9,10].

The role of colostrum in transferring immunity from ewes to lambs is crucial for the early protection of newborns against various infections, including T. gondii [7]. Colostrum, being rich in immunoglobulins, provides passive immunity to lambs, which are born with an underdeveloped immune system. Investigating the presence and transfer of IgG antibodies specific to T. gondii from ewe to lamb via colostrum can provide insights into how well the offspring are protected against toxoplasmosis [11,12]. Thus, understanding the dynamics of T. gondii infection in sheep and implementing effective control measures are critical for the sustainability of the sheep industry.

This study aims to fill the gap in understanding the correlation between IgG antibodies anti-T. gondii in the serum and colostrum of naturally infected ewes and the subsequent immunity observed in newborn lambs, ultimately contributing to improved management practices and health outcomes in sheep flocks.

2. Materials and Methods

2.1. Experimental Design

Twenty sheep breeding farms in the semi-arid region of the State of Paraíba, Brazil, were selected according to convenience (Figure 1). All the visited farms had a history of abortions and premature births. During the study, 162 blood and colostrum samples were collected from ewes and 182 samples were collected from their newborn offspring (in cases of twin births, samples were collected from both lambs).

On each farm, ewes that passed a general physical examination and were deemed healthy were selected for serological and colostral sampling. All selected ewes had recently lambed and were no more than five days postpartum. Blood samples were also collected from the respective lambs to detect anti-T. gondii antibodies and to evaluate passive immunization against the parasite.

2.2. Sample Collection

Blood was collected from each selected ewe via venipuncture of the jugular vein. Immediately after, colostrum was collected by manual milking. Simultaneously, blood samples were taken from the newborn lambs of these ewes between the second and fifth day after birth to investigate the passive transfer of anti-T. gondii IgG.

Colostrum was collected aseptically in sterile test tubes. The first three milk jets were always discarded to avoid contamination, and then approximately 5 mL of colostrum was collected from each ewe. Animals that presented with clinical mastitis were not included in this study.

The blood samples were centrifuged at 1000× g for 10 min to obtain serum for analysis. The colostrum was also centrifuged, and after this procedure, the superficial fat layer (supernatant) was discarded, leaving only the colostrum precipitate for analysis. The serum and colostrum precipitate from each animal were properly labeled and stored at −20 °C until examination.

2.3. Serological and Colostral Tests

The serum samples (from ewes and lambs) and colostrum precipitate samples were subjected to the indirect fluorescence antibody test (IFAT) for anti-T. gondii IgG, following a consistent protocol. Due to the genetic and immunogenic similarity between T. gondii and Neospora caninum, serum from all the sheep was also tested using the IFAT for anti-N. caninum IgG. This helped to ensure that positive results for T. gondii were accurate and not due to cross-reactivity with N. caninum, thereby increasing the specificity of the diagnosis.

The IFATs for anti-T. gondii IgG were performed according to Camargo [13], using an RH strain of T. gondii tachyzoites fixed on a slide, with a cutoff point of 1:64 [14]. For the IFAT for anti-N. caninum IgG, the cutoff point for blood serum and colostrum, adapted from Camillo et al. [15], was set at 1:50. The Nc-1 strain of N. caninum tachyzoites, fixed on a slide, was used as the antigen [1].

A whole-molecule sheep anti-IgG conjugate (Sigma, St. Louis, MO, USA) was used. Serum and colostrum precipitate samples that reacted at dilutions of 1:64 or greater were considered positive. Reactive serum and colostrum precipitate samples were titrated in sequential two-fold dilutions until a negative result was obtained.

2.4. Statistical Analysis

A concordance analysis was performed on the results from the IFATs between serum samples from the ewes and their offspring and the colostrum samples using the kappa coefficient [16]. The kappa values are as follows: 0.20 to 0.40 indicates reasonable agreement; 0.40 to 0.60 indicates moderate agreement; 0.60 to 0.80 indicates substantive agreement; and 0.80 to 1.00 indicates perfect diagnostic agreement. The sensitivity and specificity of antibody detection in colostrum via IFATs were calculated by comparing them with the detection of antibodies in blood serum samples. The blood serum samples from ewes and lambs were used as the standard for evaluating the sensitivity and specificity of antibody detection in colostrum [15].

3. Results

Among the 162 serological samples from ewes, 45.1% (73/162) were positive for anti-T. gondii IgG, with titers ranging from 1:64 to 1:16,384. All ewes positive for anti-T. gondii antibodies were negative for anti-N. caninum antibodies. It was observed that 5.4% (4/73) of the positive ewes had colostrum with antibody titers below 1:64. The titers in their colostrum ranged from 1:64 to 1:8192 (

Table 1. Detection and titration of anti-T. gondii IgG antibodies by indirect fluorescence antibody test (IFAT) in ewe blood serum, colostrum, and lamb serum.

Identification of the Ewes and Their Lambs	Days after Partum	Serum Titration of Ewes	Colostrum Titration	Serum Titration of Lambs
1	4	2048	256	128
2	4	256	512	64
3	3	512	64	512
9	2	512	128	<64
10	5	2048	128	64
11	2	128	256	128
12-L1	3	256	64	128
12-L2	3	256	64	64
12-L3	3	256	64	64
15-L1	3	256	64	1024
15-L2	3	256	64	128
20	2	128	64	256
21	2	64	256	1024
22-L1	2	512	512	1024
22-L2	2	512	512	2048
23	2	2048	256	4096
24	2	128	64	1024
27-L1	4	64	<64	64
27-L2	4	64	<64	64
31	3	256	256	128
33	4	256	128	2048
34	3	64	64	128
41-L1	5	128	64	512
41-L2	5	128	64	64
41-L3	5	128	64	64
42	4	256	64	1024
44	4	256	64	128
45	2	256	64	512
46-L1	3	128	256	512
46-L2	3	128	256	64
47	3	1024	64	256
48-L1	4	16,384	2048	64
48-L2	4	16,384	2048	128
50	4	128	<64	<64
51	3	128	1024	128
53-L1	2	8192	1024	512
53-L2	2	8192	1024	256
54	3	128	64	<64
55	4	512	512	64
57	4	64	512	64
58	2	2048	64	128
59	3	4096	64	64
60	4	1024	1024	256
61	2	4096	128	128
63	2	512	64	128
64	2	256	64	128
65-L1	4	4096	64	128
65-L2	4	4096	64	256
66	4	2048	64	128
67	3	8192	512	512
68	4	4096	64	64
74	3	8192	64	<64
75	4	512	64	64
79-L1	4	256	512	1024
79-L2	4	256	512	1024
80	3	4096	2048	128
81	4	256	64	128
85	2	512	64	512
86	3	256	64	2048
87	4	8192	64	4096
88	3	8192	64	8192
89	4	512	64	4096
93	3	4096	128	512
96	2	4096	512	128
101	5	2048	128	1024
105	3	512	64	2048
111	4	1024	<64	<64
113	3	64	64	512
115	4	1024	64	8192
117	2	64	64	512
118	3	128	64	256
136	2	64	64	64
141	4	64	64	64
145	4	1024	64	4096
146	5	1024	64	128
147	4	64	256	512
149	3	1024	256	256
151	3	4096	2048	256
152	4	256	256	64
154	3	128	512	256
155	3	64	512	512
156	3	512	256	512
157	5	128	256	512
161	3	256	<64	<64
162	3	8192	512	64

). Among the positive ewes, one had a high titer in its serum (1:1024), but negative results in the colostrum (<1:64; animal 111).

This study found that 74% (54/73) of the ewes with positive serum had higher titers than their colostrum, while 13.7% (10/73) of ewes had lower titers in their serum compared to their colostrum. Only 12.3% (9/73) of ewes had matching titers between serum and colostrum (Table 1).

A perfect agreement was observed between the IFAT results for ewe serum and colostrum, with a kappa coefficient of 0.950 (

Table 2. Anti-T. gondii IgG antibody detection in ewe colostrum and serum by indirect fluorescence antibody test (IFAT)—positive and negative results.

Titer of the Ewes	Positive/Analyzed Ewes (%)	Positive Colostrum/Positive Ewes (%)	Kappa
1:64	10/162 (6.2)	9/10 (90)	0.944
1:128	10/162 (6.2)	9/10 (90)	0.944
1:256	13/162 (8.0)	12/13 (92.3)	0.957
1:512	10/162 (6.2)	10/10 (100)	1.000
1:1024	7/162 (4.3)	6/7 (85.7)	0.920
1:2048	7/162 (4.3)	7/7 (100)	1.000
1:4096	9/162 (5.6)	9/9 (100)	1.000
1:8192	6/162 (3.7)	6/6 (100)	1.000
1:16,384	1/162 (0.6)	1/1 (100)	1.000
Total	73/162 (45.1)	69/73 (94.52)	0.950 1

¹ Kappa values: 0.20 to 0.40 indicates reasonable agreement; 0.40 to 0.60 indicates moderate agreement; 0.60 to 0.80 indicates substantive agreement; and 0.80 to 1.00 indicates perfect diagnostic agreement between the tests evaluated.

). The titer levels influenced the kappa coefficient, whereby ewes with low titers showed more discordance between serum and colostrum, while ewes with higher titers exhibited better kappa agreement between the two.

The kappa agreement between lamb serum and colostrum was 0.962, which is considered perfect agreement.

4. Discussion

Anti-T. gondii antibodies were found in the serum of lambs and ewes, as well as in their colostrum. This is the first study to demonstrate the kappa agreement between serum and colostrum IgG anti-T. gondii detection using IFATs on sheep. Among the positive ewes, 94.52% (69/73) also had positive colostrum. Similarly, a study by Liu et al. [17] observed a high sensitivity, reporting that 89% (57/64) of cows positive for IgG anti-T. gondii secreted milk that was also positive. Although milk was used instead of colostrum, antibodies were more frequently found in serum than in milk. The ewes with titers > 1:256 showed higher possibilities of immunizing their offspring, the ewes with a ≤1:256 serum titer presented 96.2% (51/53) positive colostrum when compared to a ≤1:128 titer, for which only 90% of the colostrum presented antibodies (18/20).

Failure to transfer anti-T. gondii antibodies was detected in 8.3% of the newborns (6/73). This failure could be attributed to the low antibody concentration in the colostrum, as all affected newborns were associated with colostrum titers ≤ 1:128, which indicates a potential compromise in passive immunization. According to Costa et al. [7], ewes with low serum titration can fail to pass enough antibodies to the colostrum and their lamb. Additionally, the lambs might have been fed milk from other females, which likely did not contain antibodies against T. gondii. One ewe, despite having a high serum titer (1:1.024), had a significantly lower titer in colostrum (<1:64), which could imply issues with antibody transfer or variability in colostrum quality.

The elevated antibody levels in some lambs, even when their dams’ colostrum titers are relatively low, support the hypothesis of vertical transmission rather than passive transfer from colostrum. Moreover, the variability in serum titers among the lambs, with some showing very high levels of antibodies, indicates that vertical transmission can lead to a significant immune response in the offspring [7,18].

A significant finding was the high kappa coefficient of 0.950, indicating perfect agreement between serum and colostrum antibody results. This high agreement highlights the reliability of colostrum IFATs as a surrogate marker for serum antibody levels in ewes. The kappa values varied with titer levels, showing better concordance in ewes with higher titers. Although diagnosis by means of IFATs demands specialized labor and takes a considerable time to perform, the high agreement between the ewes’ serum and their colostrum (0.950) and the agreement between colostrum and the newborns’ serum (0.962) could be used as a tool for the diagnosis of both lamb and dam, by performing IFATs only in the colostrum.

5. Conclusions

This study provides valuable data into the serological relationships between ewe serum and colostrum for anti-T. gondii antibodies, as well as lamb serum, with a high concordance between these three matrices. It suggests that colostrum IFATs are a reliable method for assessing maternal antibody transfer, with high-titer ewes generally providing better passive immunity to their lambs. This approach could be instrumental in managing and controlling T. gondii infections in sheep herds, ensuring the effective immunization of lambs against this parasite.