Toxoplasma gondii infection in dairy ewes: Vertical transmission and influence on milk production and reproductive performance

Microbial Pathogenesis 99 (2016) 101e105

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Toxoplasma gondii infection in dairy ewes: Vertical transmission and influence on milk production and reproductive performance
dio a, Vanderlei Klauck a, Rafael Pazinato a, Willian M. Radavelli a, Edimar Custo Anderson E. Bianchi b, Giovana Camillo c, Alfredo S. Cezar c, Fernanda F. Vogel c,
rio Ferreira a, Lenita M. Stefani a, Aleksandro S. Da Silva a, * Alexandre A. Tonin d, Roge
, SC, Brazil Department of Animal Science, UDESC Oeste, Universidade do Estado de Santa Catarina, Chapeco
, Brazil Department of Animal Science, Universidade Federal do Parana Department of Preventive Veterinary Medicine, Universidade Federal de Santa Maria, Santa Maria, RS, Brazil d Universidade do Oeste de Santa Catarina e Campus II, Xanxer^ e, SC, Brazil a

b c

a r t i c l e i n f o

a b s t r a c t

Article history: Received 23 July 2016 Received in revised form 8 August 2016 Accepted 10 August 2016 Available online 13 August 2016

The present study aimed to evaluate the transmission of toxoplasmosis (vertical and venereal) and its influence on milk production and reproductive problems of Lacaune sheep seropositives for Toxoplasma gondii. Males and females were serologically selected using indirect immunofluorescence method in three steps of the study. Step 1: In order to evaluate the influence of toxoplasmosis on milk production, the volume of milk produced by 40 sheep (22 seronegatives and 18 seropositives for T. gondii) was weekly measured throughout the lactation period. There were no significant differences between these two groups; in other words, toxoplasmosis did not affect milk production. Step 2: In order to assess T. gondii venereal transmission, five samples of semen from seropositive rams (n ¼ 5) were tested by endpoint and real time PCR with two days of interval; however, these semen samples were PCR negatives for T. gondii. Step 3: To evaluate reproductive problems, 12 seropositive animals out of a flock of 68 pregnant ewes showed signs of reproductive problems, such as abortion or fetal resorption. T. gondii transplacental transmission was evaluated on blood drawn from newborn lambs (n ¼ 41), and their respective seropositive mothers (n ¼ 30) after single, double or triple births. Serological tests showed that 65.8% of the lambs had antibodies against this protozoan, indicating a high transmission from ewe to fetus during pregnancy. Therefore, it is concluded that toxoplasmosis in sheep may impair reproduction with a high percentage of vertical transmission. © 2016 Elsevier Ltd. All rights reserved.

Keywords: Toxoplasmosis Sheep Reproductive problems

1. Introduction Toxoplasmosis is a parasitic disease of humans, wild, and domestic animals caused by Toxoplasma gondii, a cosmopolitan protozoan of the Sarcocystidae family with animals of the Felidae family as definitive hosts [1]. Toxoplasmosis is usually subclinical [2], but the most common signs in symptomatic patients are hyperthermia, dyspnea, and neurological problems. The infection can trigger severe diseases, particularly when transmitted congenitally between humans or immunocompromised individuals, causing

~o Supe* Corresponding author. Departamento de Zootecnia, Centro de Educaça
dio Zootecnia, Bairro Santo Anto ^ nio, Chapeco
, 89815-630, rior do Oeste (UDESC), Pre SC, Brazil. E-mail address: [email protected] (A.S. Da Silva). http://dx.doi.org/10.1016/j.micpath.2016.08.012 0882-4010/© 2016 Elsevier Ltd. All rights reserved.

losses on production and reproduction performance [3]. T. gondii is described as a major causative agent of abortion and in this case, the placenta is invaded by tachyzoites, resulting in mineralization and necrosis [4]; thus, the interference on fetal development may result in abortion, mummified fetus, or debilitated and weak newborns [5e10]. The prevalence of toxoplasmosis varies from region to region, suffering interference of climate and geographical factors, as well as human and animal habits [5]. Its transmission to humans and animals mainly occurs through the consumption of tissue cysts from undercooked meat, oocysts intake from pastures and food, which are all influenced by poor hygiene [11,12]. Some authors estimate that one third of the human population has antibodies against T. gondii [13]. In sheep, researchers have highlighted that the main form of transmission is by ingestion of oocysts [14]. Lopes [2]

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isolated T. gondii from testes, epididymis, seminal vesicles and prostate of sheep experimentally infected by oocysts and tachyzoites using bioassays and PCR. In another study, Lopes et al. [15] have reported similar results as Teale et al. [16], where T. gondii was isolated from seminal fluid of rams experimentally inoculated, suggesting T. gondii venereal transmission. Therefore, this study aimed to evaluate the influence of toxoplasmosis on milk production, reproductive problems, and forms of T. gondii transmission between seropositives dairy ewes. 2. Materials and methods 2.1. Animals This study was conducted in a rural property located in the Western region of Santa Catarina State, Southern Brazil. Males and females were tested on three different steps to evaluate the influence of toxoplasmosis on milk production (Step 1), possible venereal transmission (Step 2), as well as transplacental transmission and occurrence of reproductive problems (Step 3). All animals of each step were confined in collective pens (24 m2 each) with wood shaving floors and fed with silage, concentrate (50:50), and water ad libitum. T. gondii-seropositive Lacaune sheep were selected by an indirect immunofluorescence assay (IFA) searching for immunoglobulin G (IgG) anti-T. gondii in serum samples with microscope slides containing tachyzoites of T. gondii RH strain. Sera samples were previously diluted in PBS (pH 7.2) up to 1:64 (standard titration in the IFA technique for T. gondii), and incubated for 30 min at 37  C in a humidified chamber. A secondary antibody (anti-sheep-IgG conjugated to fluorescein; Sigma-Aldrich®) was added and incubated for 30 min at 37  C in a humidified chamber. Sera samples from positive and negative sheep were used as controls. Samples IFA positives at 1:64 or higher were considered positives and subjected to titration (1:128; 1:256; 1:512; and 1:1024), according to the literature [17]. Reactions with peripheral or diffuse fluorescence of tachyzoites were considered positives, as well as fluorescence with apical or polar reactions were considered negatives [17]. 2.1.1. Step 1: Influence on milk production Forty adult Lacaune females in the beginning of the lactation period with similar body weight (56.2 ± 3.9 kg), age (24.2 ± 1.1 months), and number of offsprings were divided into two groups: seropositives (n ¼ 18) and seronegatives (n ¼ 22) for T. gondii. Blood samples for serological tests were collected 7 days post-partum. The animals were confined into four collective pens (n ¼ 10), and received the same diet twice a day (silage and concentrate at proportion of 50:50). The food provision was proportionally calculated based on their body weight, therefore isoenergetic and isonitrogenous diets were prepared. Automated milking was performed twice a day (05:00 a.m. and 05:00 p.m.). The volume of milk produced was individually weighed once a week, up to seven weeks using a specific equipment (True-test®, Auckland, New Zealand). In the subsequent weeks, milk samples were collected until the end of lactation, totaling 126 days of lactation). Milk composition (fat, protein, and lactose) was determined using an infrared analyzer (Bentley 2000®) on days 7, 21, 35, 56, 84, and 126 of the experiment. 2.1.2. Step 2: Presence of T. gondii in semen In order to find T. gondii positive rams, eleven breeding males underwent serological testing by IFA. Five of them were seropositives with titers ranging from 1:128 to 1:2048. Semen collection was carried out using an artificial vagina and a collection dummy. Five semen samples were collected from each male within two days

of interval. The ejaculate was stored in tubes and frozen at 20  C. Semen samples collected from T. gondii seropositives and seronegatives animals were tested for the presence of T. gondii DNA through endpoint PCR, qPCR, and nested PCR. Genomic DNA was extracted using the Wizard Genomic DNA Purification Kit (Promega, USA), according to manufacturer's recommendations. The extracted products were evaluated for concentration and purity using the NanoDrop 1000 Spectrophotometer (Thermo Scientific, USA). DNA templates (50 ng) from each sample with absorbance ratio > 1.7 (ranging between 260/280 nm) was used. The primers used for endpoint PCR, qPCR and nested PCR (external and internal primers) were designed using the Primer Express software (Applied Biosystems, USA), and synthesized by IDT (Brazil). For nested-PCR the internal primers used were: forward 50 -TTCATTGATGCTGTCGATCGA-30 and reverse 50 -ACAAACGCCCGAGACAAAAC-30 to amplify a fragment of 100 base pairs; and the external primers were forward 50 -GAGCAGTATGATTGGGTGTCATG30 and reverse 50 -TGCTTAAGTTCAGCGGGTAACC-30 to amplify a fragment of 320 base pairs. 2.1.2.1. PCR. DNA (50 ng) extracted from each semen sample was used as template for PCR analysis. In addition, T. gondii extracted from tissue cysts of VEG strain (kept in mice) was used as positive control, and distilled water as negative control. PCR mix was composed of: 2 ml of DNA, specific buffer including magnesium chloride, DNTP, 200 nM of each primer (primer int), Platinum Taq DNA polymerase, and distilled water up to 25 ml of total volume. The reaction conditions were: denaturation at 95  C for 60s, followed by 35 cycles of 95  C for 30s, 60  C for 60s and 72  C for 2min. All the reactions were performed in a thermal cycler PTC-100TM (MJ Research Inc, USA). The PCR products were analyzed on agarose gel (1%). 2.1.2.2. qPCR. A pair of primers was used (internal primers) for the amplification of a specific T. gondii DNA fragment (100 pb). PCR mix consisted of 2 ml of DNA, 200 nM of each primer, fluorophore, and other components of SYBR® Green PCR Master Mix (Applied Biosystems, USA). The reaction conditions consisted of 40 cycles at 95  C for 30s, 60  C for 60s, and 72  C for 2min in Step One Plus Real-Time PCR System (Applied Biosystems, USA). The results were evaluated for the presence/absence of amplified specific DNA fragment according to the melting curve of each sample. 2.1.2.3. Nested-PCR. Two pairs of primers were used: an external primer for the amplification of a specific T. gondii DNA fragment (320 pb), considered the external nested-PCR fragment; as well as an internal primer for the amplification of a specific internal fragment (100 pb), containing the fragment amplified by the external primer. Thus, it was possible to assure high sensitivity and specificity in the molecular diagnosis regarding the presence/absence of the target DNA. PCR mix was composed of 2 ml of DNA template (initial concentration of 50ng/stage 1of nested-PCR) or the amplified product from stage 1 (as DNA template of stage 2 of nestedPCR), 200 nM of each primer, 2,5U of platinum TaqDNA polymerase (Invitrogen, Brazil), buffer, and distilled water up to 25 ml. The conditions for both reactions were: 40 cycles at 95  C for 30s, 60  C for 60s and 72  C for 2min in a Step One Plus Real-Time PCR System (Applied Biosystems, USA). The amplified products of the second stage from nested-PCR were assessed by electrophoresis on agarose gel (1%). 2.1.3. Step 3: Occurrence of reproduction problems and transplacental transmission Reproductive problems (abortion or fetal resorption) were evaluated in a flock of 68 pregnant ewes, considered positives (with

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at least 1:64 of titer) for T. gondii by IFA technique using blood samples collected on day 15 post artificial insemination. Pregnancy diagnosis and monitoring of sheep with age between 3 and 5 years were performed by ultrasound. Blood was also collected 30 days pre-partum from pregnant ewes to search for antibodies against T. gondii by IFA technique, followed by titration. In addition, a new blood sampling was performed on females that had abortion or fetal resorption to assess anti-T. gondii levels in serum. Pregnant ewes seropositives for T. gondii (IFA) (30 out of 68) were used to evaluate transplacental transmission. The other sheep (n ¼ 38) were not included on the vertical transmission study, since 12 ewes had reproductive disorders, and 26 ewes had their partum during the night and blood pre-colostrum samples were not collected. Thus, blood samples (2 mL) were collected from all their respective lambs delivered alive immediately after birth right before colostrum intake. Also, as controls, blood of 10 newborns from seronegative ewes were collected and analyzed for the presence of antibodies against T. gondii. 2.2. Statistical analysis The milk volume data were subjected to normality test and transformed to logarithm. The data were analyzed by t-test for comparison of means. It was considered significant when the results obtained were P < 0.05. 3. Results 3.1. Step 1: Effect of toxoplasmosis on milk production Results regarding milk production are shown in Fig. 1. There was no significant difference (P > 0.05) between groups for the volume of milk produced during the trial period. Numerically, daily and final production of seropositive animals were higher than seronegatives. Also no differences were observed between groups regarding milk composition, i.e. fat, protein, and lactose showed similar results between seropositive and seronegative ewes (P > 0.05; Table 1). 3.2. Step 2: T. gondii in the semen T. gondii DNA was not detected in semen samples analyzed by endpoint PCR, qPCR and nested PCR.

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Table 1 Chemical composition of milk from Lacaune ewes seronegatives and seropositives for Toxoplasma gondii. Variables (%)

Days of the experiment Group

7

21

35

56

84

126

P

Protein

Seronegative Seropositive

4.61 4.63

4.58 4.62

4.55 4.57

4.31 4.33

4.32 4.35

4.27 4.30

>0.05 >0.05

Lactose

Seronegative Seropositive

4.72 4.70

4.68 4.64

4.67 4.64

4.58 4.55

4.52 4.56

4.53 4.50

>0.05 >0.05

Fat

Seronegative Seropositive

7.27 7.21

7.83 7.03

7.54 6.96

7.82 7.41

7.98 7.14

8.22 8.78

>0.05 >0.05

3.3. Step 3: Transplacental transmission and reproductive problems It was observed abortion or fetal resorption in sixteen ewes (12/ 68) seropositives for T. gondii. Antibody titration of these sera samples ranged o 1:64 (n ¼ 4) and 1:256 (n ¼ 8). The other 52 sheep gave birth apparently to healthy lambs, although the ewes were seropositives for T. gondi. Thus, these animals were used to evaluate vertical transmission, as detailed below. Results related to transplacental transmission of T. gondii in seropositive ewes are shown in Table 2. Out of 41 lambs evaluated, 27 were seropositives for T. gondii, accounting for 65.8% of the vertical transmission. Results of the antibody titration for positive lambs were as follows: 12e1:64, 8e1:128, and 7e1:256. It is possible to assure, that all ewes with antibody levels of 1:512 were able to transmit the parasite to their lambs. Lambs of seronegatives ewes showed no antibodies to T. gondii. 4. Discussion T. gondii has been described as the main causative agent of abortion and other reproductive problems in sheep since 1954. These problems have a worldwide distribution with prevalence rates ranging from 6.4 to 27.2% [7,18,19]. Likewise, our study observed that abortion is related to seropositivity of ewes infected by T. gondii. The molecular biology techniques used in semen samples from seropositive rams were unable to detect T. gondii DNA, differently of what was previously reported in dogs [20], bulls [21], goats [22], boars [23], and rams [24]. Lopes et al. [24], while studying T. gondii

Fig. 1. Mean and standard deviation of daily milk production during 126 months of lactation in Lacaune ewes seronegatives and seropositives for Toxoplasma gondii. There was no statistical difference between these groups throughout the experiment.

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Table 2 Toxoplasma gondii serology: transplacental transmission from seropositive ewes to their respective lambs. Ewes

Lambs

Identification

Titer*

Number of newborns

Titer

209 159 177 221 644 915 930 905 935 834 444 914 452 934 999 816 879 894 1035 1223 937 716 766 1007 807 927 973 983 885 899

1:512 1:512 1:256 1:512 1:128 1:512 1:128 1:512 1:512 1:512 1:256 1:256 1:512 1:512 1:256 1:256 1:256 1:256 1:256 1:256 1:256 1:256 1:256 1:256 1:256 1:256 1:256 1:128 1:256 1:256

1 1 1 1 1 2 1 1 1 1 1 2 1 2 2 1 2 1 1 1 1 2 2 1 3 1 1 2 2 1

1:256 1:256 <1:64 1:64 1:64 1:64e1:64 1:64 1:128 1:128 1:256 <1:64 <1:64 e <1:64 1:64 1:64e1:64 <1:64 e <1:64 1:256 1:128e1:64 1:64 1:64 1:256 1:256 <1:64 e <1:64 <1:64 e <1:64 1:256 1:128e1:128 e 1:128 <1:64 <1:64 1:128e1:128 <1:64 e <1:64 1:64

Note: The highest dilution tested was 1:64. Therefore, samples with complete fluorescence of tachyzoites at this dilution were considered positives (i.e.  1:64). * IFA tests were performed 30 days pre-partum.

infected rams without history of reproductive disease, noticed that males with high titers (1:4096 and 1:8192) on serology, also showed PCR positive results. In our study, rams were naturally infected and showed lower titers than those found by Lopes et al. [24] after experimental infection, which may explain our negative PCR results. According to the literature, there are may ways for T. gondii transmission, such as transplacental, fecal-oral, and carnivorism [25]. Vidotto [26] described that vertical transmission occurs when the acute stage of infection coincides with pregnancy, with severe consequences to the fetus during the first third or middle of the pregnancy. Transplacental or congenital transmission is the most frequent and severe form of transmission, with natural spread in the acute or acute relapse of chronic disease during pregnancy [27]; thus, the disease spread is inevitable in flocks with the presence of positive animals. Duncanson et al. [28], while detecting T. gondii DNA from aborted lambs of a commercial sheep flock, found high levels (94%) of transplacental transmission. In lambs born alive, 42% were seropositives for T. gondii, which represents lower levels compared to those found in this study (68%). In goats, the transplacental transmission has been reported in natural and experimental situation [29,30]. These results show that this form of transmission occurs at high percentages, suggesting a generationto-generation transmission, which could cause significant losses to the sheep industry. Despite the fact that no significant differences on milk production and composition between seropositive and seronegative animals were found, we cannot rule out the interference of toxoplasmosis on the reproduction performance. According to the literature, clinical changes of toxoplasmosis in ruminants are

similar to the ones observed in cattle neosporosis [31]; however, it is not possible to relate it to the reduction on milk production, as already reported for cows infected by T. gondii [32]. Therefore, based on our results, we can conclude that milk production was not affected in T. gondii seropositive Lacaune ewes. We suggest that reproductive problems during pregnancy in these animals are related to toxoplasmosis, and that vertical transmission of T. gondii in dairy sheep has high occurrence. However, based on the titration found on serological tests of ewes 30 days pre-partum, we are unable to assure when the fetus was infected since these females could have had a reactivation of their chronic status in the beginning or late pregnancy when no blood sampling was performed. Therefore, we cannot say that venereal transmission occurred during chronic toxoplasmosis of sheep. Ethics committee This study was approved by the Animal Welfare Committee of Universidade do Estado de Santa Catarina (UDESC) under protocol number 1.16.13. References
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