Infection dynamics following experimental challenge of pigs orally dosed with different stages of two archetypal genotypes of Toxoplasma gondii.

Toxoplasma gondii is a food-borne zoonotic parasite widespread in a variety of hosts, including humans. With a majority of infections in Europe estimated to be meat-borne, pork, as one of the most consumed meats worldwide, represents a potential risk for consumers. Therefore, we aimed to investigate the progress of T. gondii infection and tissue tropism in experimentally infected pigs, using different T. gondii isolates and infectious stages, i.e. tissue cysts or oocysts. Twenty-four pigs were allocated to treatment in four groups of six, with each group inoculated orally with an estimated low dose of either 400 oocysts or 10 tissue cysts of two European T. gondii isolates, a type II and a type III isolate. The majority of pigs seroconverted two weeks post-inoculation. Pigs infected with the type III isolate had significantly higher levels of anti-T. gondii antibodies compared to those infected with the type II isolate. Histopathological exams revealed reactive hyperplasia of the lymphatic tissue of all pigs. Additionally, a selected set of nine tissues was collected during necropsy at 50 dpi from each of the remaining 22 pigs for T. gondii DNA detection by quantitative real-time PCR. A positive result was obtained in 29.8 % (59/139) of tested tissues. The brain was identified as the most frequently positive tissue in 63.6 % (14/22) of the animals. In contrast, liver samples tested negative in all animals. The highest mean parasite load, calculated by interpolating the average Cq values on the standard curve made of ten-fold serial dilutions of the genomic DNA, corresponding to 100 to 104 tachyzoites/µL, was observed in shoulder musculature with an estimated concentration of 84.4 [0.0-442.5] parasites per gram of tissue. The study highlights the variability in clinical signs and tissue distribution of T. gondii in pigs based on the combination of parasite stages and strains, with type III isolates, particularly oocysts, causing a stronger antibody response and higher tissue parasite burden. These findings suggest the need for further investigation of type III isolates to better understand their potential risks to humans.

Inactivation of Toxoplasma gondii in dry sausage and processed pork, and quantification of the pathogen in pig tissues prior to production.

Toxoplasma gondii is an important zoonotic foodborne parasite. Meat of infected animals appears to be a major source of infection in Europe. Pork is the most consumed meat in France, with dry sausages well represented. The risk of transmission via consumption of processed pork products is largely unknown, mainly since processing will affect viability but may not entirely inactivate all T. gondii parasites. We investigated the presence and concentration of T. gondii DNA in the shoulder, breast, ham, and heart of pigs orally inoculated with 1000 oocysts (n = 3) or tissue cysts (n = 3) and naturally infected pigs (n = 2), by means of magnetic capture qPCR (MC-qPCR). Muscle tissues of experimentally infected pigs were further used to evaluate the impact of manufacturing processes of dry sausages, including different concentrations of nitrates (0, 60, 120, 200 ppm), nitrites (0, 60, 120 ppm), and NaCl (0, 20, 26 g/kg), ripening (2 days at 16-24 °C) and drying (up to 30 days at 13 °C), by a combination of mouse bioassay, qPCR and MC-qPCR. DNA of T. gondii was detected in all eight pigs, including in 41.7% (10/24) of muscle samples (shoulder, breast and ham) and 87.5% (7/8) of hearts by MC-qPCR. The number of parasites per gram of tissue was estimated to be the lowest in the hams (arithmetic mean (M) = 1, standard deviation (SD) = 2) and the highest in the hearts (M = 147, SD = 233). However, the T. gondii burden estimates varied on the individual animal level, the tissue tested and the parasitic stage used for the experimental infection (oocysts or tissue cysts). Of dry sausages and processed pork, 94.4% (51/54) were positive for T. gondii by MC-qPCR or qPCR, with the mean T. gondii burden estimate equivalent to 31 parasites per gram (SD = 93). Only the untreated processed pork sample collected on the day of production was positive by mouse bioassay. The results suggest an uneven distribution of T. gondii in the tissues examined, and possibly an absence or a concentration below the detection limit in some of them. Moreover, the processing of dry sausages and processed pork with NaCl, nitrates, and nitrites has an impact on the viability of T. gondii from the first day of production. Results are valuable input for future risk assessments aiming to estimate the relative contribution of different sources of T. gondii human infections.

Evaluation of surface plasmon field-enhanced fluorescence spectroscopy for rapid measurement of progesterone concentration in bitches.

OBJECTIVE: To compare progesterone (P4) concentrations measured with surface plasmon field-enhanced fluorescence spectroscopy (SPFS) and chemiluminescence immunoassay (CLIA) in serum and plasma samples of client-owned bitches of various ages and breeds and to determine reference ranges for P4 concentrations at various stages of the estrous cycle. SAMPLES: 102 serum samples and 104 plasma samples. PROCEDURES: In experiment 1, 1 aliquot each of serum and plasma was analyzed for P4 concentration by use of SPFS incorporated in a veterinary-specific point-of-care immunologic analyzer and CLIA. In experiment 2, serum collected from bitches in various stages of the estrous cycle was analyzed for P4 concentration by use of SPFS to establish reference ranges for each stage. RESULTS: In experiment 1, P4 concentrations measured by SPFS and CLIA were highly correlated (serum, r = 0.966; plasma, r = 0.968). In experiment 2, ranges of serum basal (proestrous) P4 concentrations (n = 114) and P4 concentrations at the estimated time of ovulation (76), during pregnancy or diestrus (107), and during the prepartum period (50) measured with SPFS were 0.42 to 1.46 ng/mL, 3.69 to 7.85 ng/mL, 11.73 to 28.24 ng/mL, and 1.54 to 3.22 ng/mL, respectively. CONCLUSIONS AND CLINICAL RELEVANCE: Because serum and plasma P4 concentrations measured with SPFS were highly correlated with those measured with CLIA and ranges of serum P4 concentrations measured with SPFS for each of phase of the estrous cycle were well-defined for the large sample size, veterinarians may be able to accurately use this veterinary-specific point-of-care immunologic analyzer with SPFS methodology to determine P4 concentrations of bitches in their daily practice.

Use of a modified hair strand test to assess the antifungal activity kinetics of dog hair after a 2% climbazole shampoo application.

BACKGROUND: The "hair strand test" was first developed as a model to evaluate the antifungal activity of antidandruff shampoos. OBJECTIVE: To assess the residual activity of an antifungal shampoo on the hair shafts of dogs after a single application, followed by bathing with a physiological shampoo one month later. ANIMALS: Six beagles (two males and four females) from a research colony. METHODS: Dogs were bathed with a 2% climbazole shampoo. Hairs were collected before application of the shampoo and at scheduled intervals for 30 days after treatment. A physiological shampoo was then applied to all dogs and hairs were collected following the same schedule. The inhibition zone around the hair shafts was measured after incubation on Sabouraud's dextrose agar plates streaked with three Malassezia pachydermatis strains. RESULTS: Inhibition zones around hairs collected from dogs bathed with 2% climbazole shampoo were significantly larger than those around hairs collected before shampooing at all time points (P = 0.003). An increase in the width of the inhibition zones around climbazole treated hairs was observed following physiological shampoo on Day 30 (P = 0.005). No significant differences were observed between Malassezia pachydermatis isolates (P = 0.571). No inhibition zones were seen around the hairs of dogs bathed with physiological shampoo only. CONCLUSIONS: The modified hair strand test is useful for the assessment of residual antifungal activity on animal hairs. Use of a physiological shampoo following antifungal shampoo therapy may increase the efficacy of the antifungal product for the control of Malassezia overgrowth.

Progesterone plays a critical role in canine oocyte maturation and fertilization.

Canine oocyte maturation and fertilization take place within the oviducts under increasing plasma levels of progesterone (P4). In order to investigate the role of P4 in these processes, 51 beagle bitches were treated with the P4 receptor antagonist aglepristone at the end of proestrus and 32 females were kept untreated. Fifteen treated and 13 control bitches were inseminated at Days +1 and +2 after ovulation (Day 0). Stages of oocyte maturation and embryo development were determined after ovariectomy at different time points after ovulation. Aglepristone did not prevent ovulation but delayed the resumption of oocyte meiosis and inhibited its progression: first metaphase I (MI) stage was observed at 173 h postovulation and 39% of oocytes reached MII as late as 335 h postovulation in treated females whereas first MI occurred at 76 h and 100% of oocytes were in MII at 109 h postovulation in controls. Aglepristone extended the stay of morphologically normal oocytes within the oviducts: first signs of oocyte degeneration were observed at 335 h in treated versus 100- to 110-h postovulation in control bitches. In inseminated females, aglepristone prevented sperm progression toward the oviducts and fertilization, although motile spermatozoa were observed in the uterine tip flush and within the cranial uterine glands. A proteomic analysis of the tubal fluid from treated and control noninseminated bitches at Day +4 found evidence of 79 differential proteins potentially involved in the oocyte phenotype. In conclusion, P4 plays key roles in postovulatory canine oocyte maturation, aging, and in fertilization.

OVGP1 is expressed in the canine oviduct at the time and place of oocyte maturation and fertilization.

In the dog, oocyte maturation, fertilization, and early embryo development take place within the oviduct in the presence of increasing circulating progesterone (P4) levels. Expression of the oviduct-specific glycoprotein 1 (OVGP1), known in other species to be estrogen-dependent, was explored by real-time quantitative reverse-transcriptase PCR, Western blotting, and immunohistochemistry in oviducts from adult Beagle bitches during anestrus and at five specific time periods around ovulation: during pro-estrus before the luteinizing hormone (LH) peak (Pre-LH); after the LH peak and before ovulation (Pre-ov); and at Days 1, 4, and 7 after ovulation (n = 6 bitches per stage). Plasma estradiol-17ß (E2) and P4 were assayed at all stages. The expression of canine OVGP1 (cOVGP1) was undetectable during anestrus, increased significantly from Pre-LH to Day 1 in parallel with a decrease in plasma E2-to-P4 levels, remained high at Day 4, then decreased at Day 7 in parallel with an increase in plasma P4 levels. In contrast to other mammals, the expression of cOVGP1 was higher in the isthmus than in the ampulla at all stages. In order to explore the potential regulation of cOVGP1 expression by steroids, the 5'-flanking region of the corresponding gene was analyzed for the presence of estrogen- (ERE) and P4-response-element (PRE). An imperfect ERE and three half-ERE were found in this region, but no PREs. In conclusion, cOVGP1 is highly expressed at the time and site of oocyte maturation and fertilization, and is probably under E2 regulation. Further studies are needed to identify the potential roles of cOVGP1 in each process.

The canine oocyte: uncommon features of in vivo and in vitro maturation.

The biology of the canine oocyte is unusual compared with that of other mammalian females. The present paper reviews both in vivo and in vitro specificities of canine oocytes. Final follicular growth in the bitch is characterised by an early appearance of LH binding sites in the granulosa, a high proportion of polyovular follicles and a preovulatory luteinisation, starting at the time of the LH surge. Through follicular fluid, preovulatory oocytes are thus exposed to high levels of progesterone, as high as 1000-fold plasma concentrations. The composition of the follicular fluid is affected by the size of the female. The more specific aspect of oocyte biology in the bitch is ovulation: oocytes are expelled immature, at the Prophase I stage. Ovulatory follicles are 6-8 mm in diameter, releasing oocytes from 110 µm, with dark cytoplasm. Resumption of meiosis occurs from 48 h postovulation, MII stages appearing 48-54 h after ovulation. The mechanisms controlling such a late meiotic resumption are still unknown. Granulosa cells seem to play a central role as in other mammalian species, but not with cAMP as the principal mediator. The importance of a transient reactivation of oocyte transcription a few hours before meiotic resumption is to be explored. These specific features may contribute to the low efficiency of IVM. Only 10-20% oocytes reach the metaphase stage and suffer from a poor cytoplasmic maturation. Moreover, in vitro culture of canine oocytes is associated with a high proportion of degeneration. To date, IVM of the oocytes is the main limiting factor for the development of assisted reproductive techniques in the canine. A better knowledge of the basic physiology of folliculogenesis and the molecular mechanisms controlling oocyte meiosis resumption in this species may allow us to overcome this obstacle.

Embryo biotechnology in the dog: a review.

Canine embryos are a scarce biological material because of difficulties in collecting in vivo-produced embryos and the inability, to date, to produce canine embryos in vitro. The procedure for the transfer of in vivo-produced embryos has not been developed adequately, with only six attempts reported in the literature that have resulted in the birth of 45 puppies. In vitro, the fertilisation rate is particularly low ( approximately 10%) and the incidence of polyspermy particularly high. So far, no puppy has been obtained from an in vitro-produced embryo. In contrast, cloning of somatic cells has been used successfully over the past 4 years, with the birth of 41 puppies reported in the literature, a yield that is comparable to that for other mammalian species. Over the same period, canine embryonic stem sells and transgenic cloned dogs have been obtained. Thus, the latest reproductive technologies are further advanced than in vitro embryo production. The lack of fundamental studies on the specific features of reproductive physiology and developmental biology in the canine is regrettable in view of the increasing role of dogs in our society and of the current demand for new biological models in biomedical technology.

IGF system and ovarian folliculogenesis in dog breeds of various sizes: is there a link?

The IGF system plays a crucial role in ovarian folliculogenesis, and changes in IGF-binding protein (IGFBP) levels modulate IGF bioavailability. Data from various mammalian models suggest a link between body size, IGF1 in serum and female reproduction parameters. Among the vertebrate species, the dog exhibits the widest span in body height. Height is known to be positively correlated with the concentration of serum IGF1. In this work, the ovarian physiology of 40 bitches exhibiting a wide span of height, and breed type was investigated. IGF1, IGF2, IGFBP3, estradiol (E(2)), and progesterone concentrations in plasma and preovulatory follicular fluid were quantified. A total of 455 follicles, 2-8 mm in diameter, were recovered at the preovulatory stage, measured, and punctured. Intrafollicular levels of IGF1 were positively correlated with plasma levels, and plasma IGF1 levels were positively correlated with both bitch height and weight. The concentrations were threefold higher in large dogs compared with small dogs. A positive correlation between intrafollicular and plasmatic IGFBP3 levels and a positive correlation between plasmatic IGFBP3 levels, and both height and weight of the bitches were observed. The number of preovulatory follicles and the diameter of the three largest follicles were positively correlated with bitch height. E(2) intrafollicular concentrations were higher in preovulatory follicles from small animals than in those from large animals. In conclusion, the strong variability in height between dogs appeared to be associated with dramatic differences in IGF1, and IGFBP3 levels, in both plasma and follicular fluid. These differences were associated with significant differences in some functional aspects of ovarian follicles.

Expression of follicle-stimulating hormone and luteinising hormone binding sites in the bitch ovary during the follicular phase.

In the female dog, in contrast with most mammals, the growing follicle starts to luteinise several days before ovulation. Little is known about the physiological control of the final follicular growth in this species. In order to better understand the pituitary regulation of follicular growth, specific binding sites for FSH and LH were localised and quantified by autoradiography using [(125)I]-porcine (p) gonadotrophins on ovarian sections (7 microm) from adult Beagle bitches during the follicular phase. Follicles were analysed either before the LH surge (n = 4 bitches; n = 117 follicles) or after the LH surge and before ovulation (n = 5 bitches; n = 110 follicles). FSH binding sites were specifically and homogeneously expressed at high levels on granulosa cells of all healthy follicles from the preantral stage onwards. In contrast, LH binding sites were detected homogeneously and at high levels only on granulosa cells of follicles larger than 1 mm in diameter, including luteinised follicles. Theca binding of LH (but not FSH) was also observed, but only when using high concentrations of [(125)I]-pLH. The overall incidence of atresia was 45.8% and was dependent upon follicular diameter. Quantitative analysis of labelling showed that atretic follicles had reduced levels of both FSH and LH binding sites compared with healthy follicles. In healthy follicles, levels of both FSH and LH binding sites changed with follicle diameter. Compared with other mammals, the acquisition of LH binding on canine granulosa cells occurs in smaller sized follicles relative to the size of ovulation.

Ultrastructure of canine oocytes during in vivo maturation.

The aim of the present study was to describe the canine oocyte ultrastructural modifications during in vivo maturation, with precise reference to the timing of the LH surge and of ovulation. Twenty-five bitches were ovariectomized at specific stages between the onset of proestrus and the fifth day post-ovulation: 65 oocytes were observed by transmission electron microscopy (TEM), either before the LH surge (n = 10), between the LH surge and ovulation (n = 12) or after ovulation (n = 43). Prior to the LH surge, the oocyte nucleus had already begun its displacement to the vicinity of the oolemma and reticulated nucleoli were infrequent. The cytoplasm showed signs of immaturity (few organelles preferentially located in the cortical zone, "mitochondrial cloud", scarce cortical granules). The LH surge was immediately followed by cumulus expansion but the ovulation occurred 2 days later. Retraction of the transzonal projections and the meiotic resumption occurred after another 3 days (5 days after the LH peak). The ovulation was then followed by gradual cytoplasmic modifications. Nucleoli re-assumed a reticulated aspect around 24 hr post-ovulation. From 48 hr post-ovulation mitochondria and SER were very numerous and evenly distributed. In conclusion canine oocyte maturation began prior to the LH surge and no cytoplasmic or nuclear modifications followed immediately the LH surge and ovulation. This study suggests that two distinct signals are needed for the final in vivo maturation: one prior to the LH surge (to induce maturation) and another one, around 3 days post-ovulation (to induce meiotic resumption).

Expression of follicle stimulating hormone and luteinizing hormone receptors during follicular growth in the domestic cat ovary.

In order to better understand the pituitary regulation of follicular growth in the domestic cat, follicle stimulating hormone (FSH) and luteinizing hormone (LH) receptors (R) were localized and quantified in relation to follicle diameter and atresia using in situ ligand binding on ovarian sections. Expression of FSHR was homogeneous and restricted to follicle granulosa cells from the early antral stage onwards, whereas expression of LHR was heterogeneous on theca cells of all follicles from the early antral stage onward, and homogeneous on granulosa cells of healthy follicles larger than 800 microm in diameter and in corpora lutea. LHR were also widely expressed as heterogeneous aggregates in the ovarian interstitial tissue. Atretic follicles exhibited significantly reduced levels of both FSHR and LHR on granulosa cells, compared with healthy follicles whatever the follicular diameter, whereas levels of LHR on theca cells were lower only for atretic follicles larger than 1,600 microm in diameter. In healthy follicles, levels of FSHR and LHR in all follicular compartments increased significantly with diameter. Although generally comparable to that observed in other mammals, the expression pattern of gonadotropin receptors in the cat ovary is characterized by an early acquisition of LHR on granulosa cells of growing follicles and islets of LH binding sites in the ovarian interstitial tissue.