Detection of CWD prions in naturally infected white‑tailed deer fetuses and gestational tissues by PMCA
Detection of CWD prions in naturally infected white‑tailed deer fetuses and gestational tissues by PMCA
Francisca Bravo‑Risi1 , Paulina Soto1 , Thomas Eckland1 , Robert Dittmar2 , Santiago Ramírez1 , Celso S.G. Catumbela1 , Claudio Soto1 , Mitch Lockwood2 , Tracy Nichols3 & Rodrigo Morales1,4*
Chronic wasting disease (CWD) is a prevalent prion disease affecting cervids. CWD is thought to be transmitted through direct animal contact or by indirect exposure to contaminated environmental fomites. Other mechanisms of propagation such as vertical and maternal transmissions have also been suggested using naturally and experimentally infected animals. Here, we describe the detection of CWD prions in naturally-infected, farmed white-tailed deer (WTD) fetal tissues using the Protein Misfolding Cyclic Amplification (PMCA) technique. Prion seeding activity was identified in a variety of gestational and fetal tissues. Future studies should demonstrate if prions present in fetuses are at sufficient quantities to cause CWD after birth. This data confirms previous findings in other animal species and furthers vertical transmission as a relevant mechanism of CWD dissemination. Chronic wasting disease (CWD) is a prion disease affecting several cervid species1–3 . CWD clinical signs (including lack of fear of people, polydipsia, polyurea, weight loss, progressive wasting/weakness, and others) are due to the accumulation of neurotoxic misfolded forms of the prion protein (PrPSc) in specific areas of the brain1,3 . PrPSc aggregates are also present in several peripheral tissues, including lymphoid tissues, peripheral nerves, muscle, blood and skin, among others4–7 . The presence of prion infectivity in peripheral tissues is considerably lower compared to the brain4,8 . Moreover, PrPSc aggregates are the infectious particles causing CWD9 .
CWD can reach high incidence in certain populations. Extensive evidence demonstrates that naïve animals are infected through direct contact with diseased animals or by indirect exposure to contaminated fomites9 . CWD prions are known to exist in low quantities in excreta10–12. However, the continuous release of infectious prion particles during most of the CWD prion incubation period and clinical disease make them relevant for disease transmission13,14. The latter is strongly supported by the fact that prions have high affinity for certain natural and manmade materials (e.g., soil, plants, stainless steel, polypropylene, among others)15–17, and are resistant to degradation18,19. Nevertheless, other possible routes of transmission, such as sexual and vertical routes, still present questions.
The potential for sexual and maternal routes in prion disease transmission has been studied in different systems, including naturally or experimentally infected sheep20–22, goats23,24, cattle25,26, and rodent models27. Many experiments in sheep, goats and Syrian hamsters suggest that sexual contact is not an efficient mean to transmit prion disease27,28. However, PrPSc has been identified in several reproductive and gestation-related tissues such as testes, ovaries, uterus, placenta and amniotic fuid21,29,30. In addition, progeny from infected deer, sheep and cattle are known to have increased risks to develop prion disease26,31,32. Abnormal accumulation of prion protein, seeding activity and/or prion infectivity have been identified in fetal tissues from sheep33 and elk30. Mother-to-offspring prion transmission appears to be prion-strain specific as evidence in other animal species including humans, Syrian hamsters and sheep infected with the classical bovine spongiform encephalopathy (BSE) agent show that progeny from infected females at the moment of gestation do not develop prion disease in the longterm22,27. For the specific case of CWD, previous reports demonstrated the presence of PrPSc in gestational tissues of naturally infected elk30 and experimentally-infected muntjac deer29. In both cases, prion identification using either the Protein Misfolding Cyclic Amplification (PMCA) technique, or immunohistochemistry (IHC) provided convincing evidence of prion accumulation in fetal tissues. Nevertheless, whether this occurs in naturally infected white-tailed deer (WTD) is still unknown.
Here, we report the presence of seeding competent CWD prions in fetal tissues collected from naturally prion-infected farmed WTD does using PMCA. The results presented in this article confirm the presence of CWD prions in fetal tissues from naturally infected farmed WTD dams suggesting that CWD could be transferred from mother to offspring.
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Discussion
CWD is rapidly expanding in both captive and wild cervid populations. While direct animal contact and environmental contamination provide reasonable explanations on how this disease is transmitted, evidence involving fetal infection and maternal exposure suggest that these routes may be relevant for disease transmission. Offspring from scrapie-infected sheep has been described as being at increased risk of developing prion disease32. Similar outcomes have been described for farmed elk41 and experimentally infected muntjac deer31. Relevant evidence supporting maternal-offspring CWD transmission include prion seeding activity identified in placenta and gestational fluids from pregnant elk and muntjac deer29,30. Importantly, prion detection has been identified in fetal tissues from elk30. Controlled experimental conditions in muntjac deer demonstrate that mother-to-offspring transmission is possible for CWD31. Our results show that fetal tissues collected from naturally infected CWD positive asymptomatic farmed WTD females contain seeding competent prions. This suggests that mother-to-offspring prion transmission is a common feature of CWD across different cervid species.
In this report, we communicate the screening of 19 fetal and gestational tissues and fluids for the detection of CWD prions. Relevant CWD positive fetal tissues include liver, kidney, and lymphoid and reproductive tissues. The case of liver and kidney is interesting, as prion accumulation in these tissues is not observed by IHC in adult CWD-symptomatic animals5 . The presence of CWD prions in fetuses’ sexual tissues is also interesting, especially considering our previous report showing that prion seeding activity is present in the testes of CWD-infected WTD bucks only at the late pre-symptomatic stages35. On the contrary, the identification of CWD prions in a large proportion of lymphoid tissues is in alignment with the expected pathophysiology of prions observed in adult animals2 . This finding suggests that the tropism of infectious prions in lymphoid organs occurs even at fetal stages. However, the results presented in this article do not allow us to conclude whether CWD prions present in fetal tissues came from the mothers through circulation or were generated de novo in the fetuses. The poor detection of CWD prions in fetal brains strongly supports the idea that neuroinvasion (ergo, prion replication) does not occur at fetal stages.
PMCA can detect prions at sub-infectious levels34,42–44 and CWD prion amplification by PMCA is able to catch sub-infectious PrPSc quantities in the first round6,35. Whether CWD prions present in fetal tissues exist in quantities large enough to induce clinical CWD after birth cannot be concluded from our results. Previous results in goats show that embryo transfer from infected to naïve females failed to transmit prion disease to ofspring28, suggesting that if prions in sheep and goat embryos contain prions, they are present in sub-infectious quantities. Nevertheless, it is important to acknowledge that embryos described in those studies were exposed to a prion-infected environment for a restricted time, and either prion absorption and replication by embryos may be limited. The latter assumption is supported by the fact that recipient females were not infected28. Nonetheless, similar studies in sheep demonstrated that in utero prion transmission is possible45. The presence of prion infectivity in mammary glands, colostrum and milk of sheep suggest that transmission can also occur after birth46–49.
Future studies detecting prions in mammary glands and milk of deer does will help us to evaluate the different possible scenarios in which CWD can be transmitted from mother to offspring (i.e., in utero vs. milking/nursing). Research in this area is relevant considering that wild WTD CWD-positive does seems more likely to be parents compared to their CWD-negative counterparts50.
The results presented in this study show that CWD prions exist in WTD fetuses from naturally infected does. Whether prions in fetal tissues are enough to sustain infectivity after birth, as well as descriptions of the mechanisms governing mother-to-offspring CWD transmission in cervids, should be clarified in future studies. These studies should include the screening of larger number of samples collected from wild and farmed animals affected by different strains of CWD prions, bioassays in susceptible mice to measure infectivity titers, and controlled experiments using pregnant/CWD-infected WTD females.
Received: 21 June 2021; Accepted: 30 August 2021 Published online 15 September 2021
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Terry S. Singeltary Sr.
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