Direct detection and quantification of Toxoplasma gondii in meat samples from feral raccoons (Procyon lotor) in Germany by magnetic-capture real-time PCR.

Because the number of wild raccoons in Germany is increasing constantly, it appears to be economic reasonable to use their meat as food. For this purpose, it is essential to generate data regarding the pathogen load of the meat to be consumed and handled. It is known that raccoons, particularly in Germany, show a high seroprevalence of Toxoplasma gondii. Because serological data only indicates contact of a host to a parasite additional direct detection is needed to prove presence of parasitic stages in particular tissues. Therefore, a total of 150 samples from raccoons with known serostatus were tested and quantified using magnetic-capture real-time PCR for Toxoplasma gondii. As it represents potentially consumption-relevant parts of raccoons, meat from forelimb and hindlimb was examined. Samples were stratified into three groups based on the animals' serostatus (each 50 negative, low positive, and high positive). All samples from seronegative animals were found negative by MC-PCR as well. In a total of 56 meat samples from 100 seropositive animals, T. gondii DNA was detected. Statistically significant more samples were positive by MC-PCR in the high positive than in the low positive serostatus group (38/50 vs. 18/50, p < 0.0001). Furthermore, samples from the former group were also found to have statistically significant higher DNA equivalent values compared to samples from the low positive serostatus group (p < 0.0001). These results suggest that meat from seropositive raccoons may contain considerable numbers of T. gondii presenting a potential public health risk for humans whilst handling and consumption.

Toxoplasma gondii in raccoons (Procyon lotor) in Germany: a serosurvey based on meat juice.

Toxoplasma gondii seroprevalence was determined in meat juice samples of 820 free-living raccoons from Germany. The animals were collected between December 2017 and April 2021. Using a commercial enzyme linked immunosorbent assay (ELISA), the overall seroprevalence was found to be 48.5%. Statistical analysis revealed significant seroprevalence differences between seasons, sex, and weight of analysed raccoons. The prevalence in late winter/spring (57.7%) was significantly higher than in autumn (38.4%) (p < 0.0003). Male raccoons (50.5%) were more often seropositive than females (41.0%) (p = 0.028). Increasing animal weight had a significant impact on the relative probability of a positive serostatus (odds ratio: 1.783, p < 0.0001). Furthermore, we found regional differences in seroprevalence, but there was no statistically significant difference resulting from animal age, degree of habitat urbanization and hunting year. Meat juice is a suitable medium for serological surveys for T. gondii in meat producing animals, as sampling is even possible after slaughter or during meat inspection when blood is no longer available. The observed high seroprevalence indicates that T. gondii infection is widespread among the German raccoon population providing a potentially relevant source of T. gondii transmission to humans upon consumption or handling of animal products.

Unique clockwork in photoreceptor of rat.

In mammals, the retina contains a clock system that oscillates independently of the master clock in the suprachiasmatic nucleus and allows the retina to anticipate and to adapt to the sustained daily changes in ambient illumination. Using a combination of laser capture micro-dissection and quantitative PCR in the present study, the clockwork of mammalian photoreceptors has been recorded. The transcript amounts of the core clock genes Clock, Bmal1, Period1 (Per1), Per3, Cryptochrome2, and Casein kinase Iε in photoreceptors of rat retina have been found to undergo daily changes. Clock and Bmal1 peak with Per1 and Per3 around dark onset, whereas Casein kinase Iε and Cryptochrome2 peak at night. As shown for Clock, Per1, and Casein kinase Iε, the oscillation of transcript amounts results in daily changes of the protein products. The in-phase oscillation of Clock/Bmal1 with Pers and the rhythmic expression of Casein kinase Iε do not occur in molecular clocks of other tissues including the suprachiasmatic nucleus. Therefore, the findings presented suggest that the photoreceptor clock is unique not only in its position outside the clock hierarchy mastered by the suprachiasmatic nucleus, but also with regard to the intrinsic rhythmic properties of its molecular components.

Active zone proteins are dynamically associated with synaptic ribbons in rat pinealocytes.

Synaptic ribbons (SRs) are prominent organelles that are abundant in the ribbon synapses of sensory neurons where they represent a specialization of the cytomatrix at the active zone (CAZ). SRs occur not only in neurons, but also in neuroendocrine pinealocytes where their function is still obscure. In this study, we report that pinealocyte SRs are associated with CAZ proteins such as Bassoon, Piccolo, CtBP1, Munc13-1, and the motorprotein KIF3A and, therefore, consist of a protein complex that resembles the ribbon complex of retinal and other sensory ribbon synapses. The pinealocyte ribbon complex is biochemically dynamic. Its protein composition changes in favor of Bassoon, Piccolo, and Munc13-1 at night and in favor of KIF3A during the day, whereas CtBP1 is equally present during the night and day. The diurnal dynamics of the ribbon complex persist under constant darkness and decrease after stimulus deprivation of the pineal gland by constant light. Our findings indicate that neuroendocrine pinealocytes possess a protein complex that resembles the CAZ of ribbon synapses in sensory organs and whose dynamics are under circadian regulation.

Daily oscillation of gene expression in the retina is phase-advanced with respect to the pineal gland.

The photoreceptive retina and the non-photoreceptive pineal gland are components of the circadian and the melatonin forming system in mammals. To contribute to our understanding of the functional integrity of the circadian system and the melatonin forming system we have compared the daily oscillation of the two tissues under various seasonal lighting conditions. For this purpose, the 24-h profiles of the expression of the genes coding for arylalkylamine N-acetyltransferase (AA-NAT), nerve growth factor inducible gene-A (NGFI-A), nerve growth factor inducible gene-B (NGFI-B), retinoic acid related orphan receptor beta (RORbeta), dopamine D4 receptor, and period2 (Per2) have been simultaneously recorded in the retina and the pineal gland of rats under short day (light/dark 8:16) and long day (light/dark 16:8) conditions. We have found that the cyclical patterns of all genes are phase-advanced in the retina, often with a lengthened temporal interval under short day conditions. In both tissues, the AA-NAT gene expression represents an indication of the output of the relevant pacemakers. The temporal phasing in the AA-NAT transcript amount between the retina and the pineal gland is retained under constant darkness suggesting that the intrinsic self-cycling clock of the retina oscillates in a phase-advanced manner with respect to the self-cycling clock in the suprachiasmatic nucleus, which controls the pineal gland. We therefore conclude that daily rhythms in gene expression in the retina are phase-advanced with respect to the pineal gland, and that the same temporal relationship appears to be valid for the self-cycling clocks influencing the tissues.

Influence of photoperiodic history on clock genes and the circadian pacemaker in the rat retina.

The influence of seasonal lighting conditions on expression of clock genes and the circadian pacemaker was investigated in the rat retina. For this purpose, the 24-h profiles of nine clock genes (bmal1, clock, per1, per2, per3, dec1, dec2, cry1 and cry 2) and the arylalkylamine N-acetyltransferase gene as an indicator of the circadian pacemaker output were compared between light-dark periods of 8 : 16 and 16 : 8 h. The photoperiod influenced the daily patterns of the amount of transcript for per1, per3, dec2 and arylalkylamine N-acetyltransferase. This indicates that photoperiodic information modulates clock gene expression in addition to the circadian pacemaker of the retina. Under constant darkness, photoperiod-dependent changes in the daily profile of the level of transcript persisted for the arylalkylamine N-acetyltransferase gene but not for any of the clock genes. Hence, quantitative expression of each clock gene is influenced by the photoperiod only under the acute light-dark cycle, whereas the pacemaker is capable of storing photoperiodic information from past cycles.

The photoperiod entrains the molecular clock of the rat pineal.

The suprachiasmatic nucleus-pineal system acts as a neuroendocrine transducer of seasonal changes in the photoperiod by regulating melatonin formation. In the present study, we have investigated the extent to which the photoperiod entrains the nonself-cycling oscillator in the Sprague-Dawley rat pineal. For this purpose, the 24-h expression of nine clock genes (bmal1, clock, per1, per2, per3, cry1, cry2, dec1 and dec2) and the aa-nat gene was monitored under light-dark 8 : 16 and light-dark 16 : 8 in the rat pineal by using real-time RT-PCR. The 24-h pattern of the expression of only per1, dec2 and aa-nat genes was affected by photoperiod. In comparison with the short photoperiod, the duration of elevated expression under the long photoperiod was elongated for per1 and shortened for dec2 and aa-nat. For each of the genes, photoperiod-dependent variations partly persisted under constant darkness. Therefore, the pineal clockwork appears to memorize the photoperiod of prior entrained cycles. The findings of the present study indicate that the nonself-cycling oscillator of the rat pineal is entrained by photoperiodic information and therefore that it participates in seasonal timekeeping.

Circadian gene expression patterns of melanopsin and pinopsin in the chick pineal gland.

The directly light-sensitive chick pineal gland contains at least two photopigments. Pinopsin seems to mediate the acute inhibitory effect of light on melatonin synthesis, whereas melanopsin may act by phase-shifting the intrapineal circadian clock. In the present study we have investigated, by means of quantitative RT-PCR, the daily rhythm of photopigment gene expression as monitored by mRNA levels. Under a 12-h light/12-h dark cycle, the mRNA levels of both pigments were 5-fold higher in the transitional phase from light to dark than at night, both in vivo and in vitro. Under constant darkness in vivo and in vitro, the peak of pinopsin mRNA levels was attenuated, whereas that of melanopsin was not. Thus, whereas the daily rhythm of pinopsin gene expression is dually regulated by light plus the intrapineal circadian oscillator, that of melanopsin appears to depend solely on the oscillator.

Arylalkylamine N-acetyltransferase gene expression in retina and pineal gland of rats under various photoperiods.

The present study examines how the circadian oscillators in the retina and the suprachiasmatic nucleus (SCN) respond to changes in photoperiod. Arylalkylamine N-acetyltransferase (aa-nat) gene expression studied by quantitative RT-PCR revealed that in adult Sprague-Dawley rats kept under different light-dark (LD) cycles for two weeks the temporal pattern of AA-NAT mRNA expression was identical in retina and pineal gland. In both tissues, the time span between the onset of darkness and the nocturnal rise in AA-NAT mRNA expression was 3 h under LD 20:4, 6 h under LD 12:12, and 15 h under LD 4:20. As aa-nat expression in the pineal gland is regulated by the circadian oscillator in SCN, the results suggest that the photoperiodic differences accompanying the seasons of the year are imprinted in more than one oscillator and that this may accentuate the important message regarding 'time of year.'

Rat pineal arylalkylamine-N-acetyltransferase: cyclic AMP inducibility of its gene depends on prior entrained photoperiod.

The nocturnal biosynthesis of melatonin in the rat pineal depends on strongly enhanced expression of the enzyme N-acetyltransferase [arylalkylamine N-acetyltransferase (AA-NAT); EC 2.3.1.87]. AA-NAT transcription is stimulated during darkness by adrenergic inputs to the pineal from the suprachiasmatic nucleus (SCN). Nocturnal activation of the AA-NAT promotor following stimulation of pinealocyte adrenoceptors involves cAMP-dependent stimulation of protein kinase A (PKA). The nocturnal rise in AA-NAT depends on the lighting conditions. As compared with light/dark (LD) 12:12, the delay between dark onset and the nocturnal rise in AA-NAT is shortened under long photoperiods and prolonged under short photoperiods. Here, we report that the rapidity of nocturnal AA-NAT induction depends on cAMP inducibility of the gene. Accordingly, cAMP produces a strong AA-NAT response in pineals obtained from rats housed under long photoperiods and a weak AA-NAT response under short photoperiods. Changes in AA-NAT inducibility are fully developed not earlier than after seven cycles. This observation suggests that long-term changes in the photoperiod are necessary to achieve full adjustment of cAMP inducibility of the gene. A direct relationship was found between cAMP-dependent AA-NAT inducibility and the pineal protein kinase A (PKA) activity. As compared to LD 12:12, PKA activity was increased under LD 20:4 and attenuated under LD 4:20. On the basis of the present findings, we suggest that the photoperiod determines the effectiveness of nocturnal AA-NAT induction by long-term modulation of the intrapineal pathway that transmits the cAMP signal to the AA-NAT gene.