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.

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.

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.