Detection of Toxoplasma gondii in the milk of experimentally infected Wistar female rats

The milk of experimentally infected rats was investigated for the presence and possible transmission of Toxoplasma gondii. Wistar (Rattus norvegicus) female rats were divided into three groups and orally inoculated with bradyzoites. Polymerase chain reaction (PCR) was employed to detect the parasite in the milk. Transmission to the offspring was verified by indirect immunofluorescence antibody test (IFAT), modified agglutination test (MAT), bioassay tests and PCR. Rat milk samples were PCR-positive, pups were serumreactive to T. gondii and tissue samples also presented positive DNA results through PCR.


INTRODUCTION
Toxoplasmosis, a significant zoonosis, is caused by Toxoplasma gondii, whose transmission to humans can be due to consumption of infected milk (1)(2)(3).The milk of affected animals contains tachyzoites, which facilitates the transmission to the offspring (4).Riemann et al. (5) reported a case of T. gondii infection in children who probably contracted the disease by ingesting unpasteurized goat milk.
Among the studies on vertical transmission, only a few have reported the presence of tachyzoites in the milk of different species including sheep, goats, cattle and experimentally infected mice (6)(7)(8).Several aspects of the mechanisms responsible for toxoplasmic infection have been elucidated by using experimental models (9,10).
Rats are the animals most often employed in these works (11).Thus, the current study evaluated whether T. gondii was present in and transmitted through the milk of experimentally infected rats.
The experiment included 21 Wistar (Rattus norvegicus) rats, 18 were females and three were males used only in breeding.All were aged between 100 and 120 days and were seronegative for toxoplasmosis, which was confirmed before the beginning of the experiment by IFAT, according to Camargo (12), and by MAT, as described by Desmonts and Remington (13).In both assays, the titer 16 was considered positive.
The experiment started only after the negative result had been obtained.
Females were divided into three groups: G1, G2 and G3.The G1 and G2 animals were inoculated by oral gavage with 10 4 bradyzoites of the strain BTU4, named after the city, Botucatu, where it was isolated and classified as genotype I. G1 and G2 rats were observed for 60 days after inoculation.During this period, serological assays were carried out fortnightly by collecting blood samples from animals through retroorbital puncture with capillary tubes.Rats of the three groups mated and were daily observed until labor.At the 60 th day, G3 animals were infected as previously described and G2 rats were subjected to immunosuppression with dexamethasone (2.5 mg/kg/day) for 15 days, following the protocol of Djurkovic-Djakovic and Milenkovic (14) adapted for the subcutaneous route.
To detect the parasite in the milk, samples were collected six times during the first 15 lactation days and submitted to PCR.Collections were carried out on alternate days until the 3 rd collection and every two days from the 4 th collection to the 15 th lactation day.To facilitate the collection of milky secretion, 1 mL (5 UI) of oxytocin was intraperitoneally injected into rats that were then milked through manual breast Costa VM and Langoni H. Detection of Toxoplasma gondii in the milk of experimentally infected Wistar female rats.J Venom Anim Toxins incl Trop Dis.2010;16(2):370 massage using a disposable sterile Pasteur pipette to aspirate approximately 300 μL of milk (15).

T. gondii transmission to the offspring was evaluated through analysis of blood
samples from pups by IFAT and MAT; bioassay -which consisted of inoculating Swiss mice with a pool of brain, lung, liver, striated skeletal muscle, tongue and diaphragm tissue samples from each litter -and by individual PCR of each of these tissue fragments.Additionally, the same types of tissue analyzed in the dams, including mammary, were collected from the offspring and individually subjected to PCR.
The pool of organs from each litter was divided into two parts: the first was digested in pepsin acid solution, according to Dubey (16), and the second was processed in natura.Both samples (digested and in natura) were subcutaneously inoculated into three Swiss mice, aged 30 days, which were observed for a 60-day period.Then, serology was performed by IFAT and MAT or, in case of death, peritoneal fluid was collected to verify the presence of tachyzoites.
DNA from milk and tissue samples was extracted by the scalable method using GFX® Genomic Blood DNA Purification kit (GE Healthcare/Life Sciences, USA) and purified with Illustra® Blood GenomicPrep Mini Spin kit (GE Healthcare/Life Sciences, USA).To verify the sensitivity of the assay, the detection threshold was determined in each tissue, by detecting variable concentrations of tachyzoites according to the milk and studied tissue.To identify T. gondii DNA in the samples, PCR was carried out by the B1 gene detection protocol of Homan et al. (17), and products were visualized through horizontal electrophoresis in 2% agar gel.
The GFX® Genomic Blood DNA Purification kit (GE Healthcare/Life Sciences, USA) permitted the detection of up to 1.5 x 10 1 tachyzoites/mL in pure suspension of peritoneal exudate and 1.5 x 10 2 tachyzoites/mL in milk samples contaminated with exudate.Also, the Illustra® Blood GenomicPrep Mini Spin Purification kit (GE Healthcare/Life Sciences, USA) allowed the detection of up to 1.5 x 10 1 tachyzoites/mL in pure exudate rich in tachyzoites and brain suspension samples; 1.5 x 10 2 tachyzoites/mL in lung, tongue and diaphragm samples; and 1.5 x 10 3 tachyzoites/mL in liver, heart, striated skeletal muscle and mammary gland samples of female rats.
For each extraction sequence and milk and tissue sample analysis through PCR, positive and negative controls were used.Positive controls comprised milk samples Costa VM and Langoni H. Detection of Toxoplasma gondii in the milk of experimentally infected Wistar female rats.J Venom Anim Toxins incl Trop Dis.2010;16(2):371 from rats infected with T. gondii at the concentration of 2.0 x 10 2 tachyzoites/mL and tissue samples at the concentrations of 2.0 x 10 2 (brain, lung and tongue suspensions) and 2.0 x 10 3 tachyzoites/mL (heart, striated skeletal muscle, liver, diaphragm and mammary gland samples).Ultrapure water was used as negative control.
Litters were reduced to six pups per dam and the remaining young animals were euthanized after 21 days, together with their mothers.A chamber saturated with isoflurane vapor was used for euthanasia (18).Ketamine hydrochloride (25 mg/kg/day) was intramuscularly injected to anesthetize the animals for blood and milk collection.
Three milk samples were PCR-positive, two on the 1 st and 5 th collection days from rat 1, of G1, and one on the 1 st day post-infection from rat 3 of G3.Serology of litters from all G1 rats indicated seroconversion; however, bioassays were negative.In G3, four pups of dam 3 seroconverted and tested positive by bioassay tests (mice inoculated with a pool of their tissues).Two pups of rat 3 and 4 also presented positive results in bioassay tests (Table 1).In G2, three dams and all litters died.The median survival was 13 days for adult rats and nine days for their offspring.Frequently observed clinical alterations included prostration and the loss of fur and weight.The peritoneal exudates from rats that died did not have tachyzoites.In this this group, serology, bioassay and PCR could not be performed in neonates.
Drugs are easily excreted through breast milk during the first days of lactation (17).It is believed that death of newborns may be due to high concentration of drugs in milk and their low capacity to biotransform them.Similarly, the immaturity of the immune system and the prolonged dexamethasone effects on the hypothalamic-pituitaryadrenal axis, resulting in reductions of antibody production and immunity, may have also contributed to early death of dams and their pups (19,20).
Among the sample sources, only skeletal muscle from inoculated rats, of all three groups, presented T. gondii DNA, with 66.66% positivity.By PCR, pup 3 from dam 1 of G1 had positive liver and skeletal muscle samples, and pup 5 showed positivity only in the liver.In this same group, dam 2 had a pup that presented positive lung samples.
This study demonstrated that T. gondii was found in the milk of experimentally infected rats, both in chronic and acute infections, which is similar to the findings of Powell et al. (21).These authors also identified by PCR T. gondii DNA in the milk of cats experimentally infected at different gestational stages with cysts.In the present study, the milk presented intermittent parasite elimination, corroborating the results of Pettersen (7).Ruchman and Fowler (22) detected the parasite in the blood of rats intraperitoneally inoculated with tachyzoites of an RH strain from four hours to 16 days after inoculation.Bonametti et al. (23) reported a case of T. gondii transmission by a mother who had contracted primo-infection by ingesting raw goat meat with cysts and possibly transmitted the parasite to the newborn through breastfeeding.
In the present study, Toxoplasma gondii was detected in the milk of experimentally infected Wistar female rats.Those are important factors that require further studies for clinical evaluation in children, since this disease tend to be highly severe in this age group, which may represent a public health problem when mothers are infected after birth.

Table 1 .
Distribution of positive and negative pups according to the diagnostic technique and experimental group