Bayesian diagnostic test evaluation and true prevalence estimation of mycoplasma bovis in dairy herds.

The true prevalence of dairy cattle herds with M. bovis infections in the Netherlands is unknown. Previous attempts to estimate prevalences were hampered by the absence of a diagnostic serological test that was validated under field conditions. This study estimated sensitivity and specificity of two commercial serum ELISAs and the true M. bovis herd prevalence using different Bayesian latent class models. A total of 7305 serum samples from 415 randomly chosen dairy herds were collected in fall/winter 2019 and investigated for presence of antibodies against M. bovis using the BIO-K-260 ELISA from Bio-X. Serum samples from 100 of these herds were also tested with a second ELISA, from IDvet. A Bayesian latent class model using the paired test results estimated a sensitivity of 14.1% (95% Bayesian probability interval (BPI): 11.6-16.7%) for the Bio-X ELISA and a specificity of 97.2% (95% BPI: 95.9-98.4%). Sensitivity and specificity for the IDvet ELISA were estimated at 92.5% (95% BPI: 88.3-96.5%) and 99.3% (95% BPI: 98.7-99.8%), respectively. Also, Bio-X ELISA sensitivity was considerably higher with data from calves only and with data from a selection of herds with a clinical outbreak, whereas the IDvet ELISA sensitivity was fairly constant under these conditions. These differences in test sensitivity is expected to be related to an effect of time since infection. A second Bayesian model, applied on test results of all 415 herds, estimated a true herd prevalence of 69.9% (95% BPI: 62.7-77.6%), suggesting M. bovis in endemic amongst dairy cattle herds in the Netherlands. To what extent seropositive herds have experienced a clinical outbreak needs further investigation.

Effectiveness of the Leptospira Hardjo Control Programme and Detection of New Infections in Dairy Cattle in The Netherlands.

Since 2005, a mandatory L. Hardjo control programme (LHCP) has been in place for Dutch dairy herds. Almost 100 percent of dairy farms participate and have an L. Hardjo-free status. In 2020 and 2021, the number of outbreaks seemed to increase as compared to the previous years. In this study, we evaluated the effectiveness of the national LHCP in the Netherlands during 2017-2021. Cases of new infections in herds with an L. Hardjo-free status in the LHCP were described, including the role of risk factors for the introduction. Both the percentage of dairy herds with an L. Hardjo-free status that purchased cattle from herds without a free status and the number of purchased cattle increased over the years. A between-herd cluster evaluation showed that between 2017 and 2021, a suspected infection was detected 144 times in 120 dairy herds. In 26 cases (26 herds, 0.2%) new infections were identified, including within-herd transmission. No infection clusters were identified, indicating that infections never led to local transmission between dairy herds. The introduction of cattle from non-free herds appeared to be the cause of all L. hardjo infections in herds participating in the LHCP. Therefore, the national LHCP seems to be highly effective in the control of infections in dairy herds.

Loss of Caprine Arthritis Encephalitis Virus (CAEV) Herd Accreditation: Characteristics, Diagnostic Approach, and Specific Follow-Up Scenarios on Large Dairy Goat Farms.

The retrovirus causing caprine arthritis encephalitis (CAE), a slowly progressive inflammatory disease in goats, belongs to the group of small ruminant lentiviruses (SRLVs) which cause lifelong infections that ought to be avoided for animal welfare as well as economic reasons. SRLV accreditation has been in place for forty years in The Netherlands and is based on the screening of small ruminant sera for specific antibodies. This paper evaluates 38 dairy goat herds that lost CAEV accreditation between 2012 and 2022. The characteristics of these herds are discussed, and specific follow-up scenarios, depending on desired goals, are introduced. The herd size of the participating herds varies from approximately 400 to 4600 adult dairy goats. The larger herds tended to be more prone to lose herd accreditation and had more difficulties regaining accreditation. Possible routes of introduction are lined up. The Royal GD's tailor-made approach and advice to support livestock farmers with herds that have lost CAE accreditation are discussed in detail. Specific emphasis is placed on the strategic deployment of various diagnostic tests (such as antibody ELISAs and PCR) in different media, such as (pooled) sera, (bulk)milk and tissue samples. Special attention is paid to the added value of retrospective bulk milk testing or the specific testing of groups based on housing and management, which enables the investigation of the moment of viral introduction and route of transmission into a herd. Furthermore, the prospective implementation of bulk milk and strategic pooled milk sample testing in the Dutch SRLV accreditation programs intensifies surveillance and enables the taking of swift action to prevent further transmission within and between herds. An appeal is made to share experiences to improve programs collectively, and to start research into the underlying mechanisms.

Monitoring and Surveillance of Small Ruminant Health in The Netherlands.

In contemporary society and modern livestock farming, a monitoring and surveillance system for animal health has become indispensable. In addition to obligations arising from European regulations regarding monitoring and surveillance of animal diseases, The Netherlands developed a voluntary system for the monitoring and surveillance of small ruminant health. This system aims for (1) early detection of outbreaks of designated animal diseases, (2) early detection of yet unknown disease conditions, and (3) insight into trends and developments. To meet these objectives, a system is in place based on four main surveillance components, namely a consultancy helpdesk, diagnostic services, multiple networks, and an annual data analysis. This paper describes the current system and its ongoing development and gives an impression of nearly twenty years of performance by providing a general overview of key findings and three elaborated examples of notable disease outbreaks. Results indicate that the current system has added value to the detection of various (re)emerging and new diseases. Nevertheless, animal health monitoring and surveillance require a flexible approach that is able to keep pace with changes and developments within the industry. Therefore, monitoring and surveillance systems should be continuously adapted and improved using new techniques and insights.

Predicting Positive ELISA Results in Dairy Herds with a Preferred Status in a Paratuberculosis Control Program.

Dairy herds participating in the Dutch milk quality assurance program for paratuberculosis are assigned a herd status on the basis of herd examinations by ELISA of individual serum or milk samples, followed by an optional confirmatory fecal PCR. Test-negative herds are assigned Status A; the surveillance of these herds consists of biennial herd examinations. Farmers falsely believing that their Status A herds are Map-free may inadvertently refrain from preventive measures. Therefore, we aimed to develop a predictive model to alert Status A farmers at increased risk of future positive ELISA results. Using data of 8566 dairy herds with Status A in January 2016, two logistic regression models were built, with the probabilities of ≥1 or ≥2 positive samples from January 2017-June 2019 as dependent variables, and province, soil type, herd size, proportion of cattle born elsewhere, time since previous positive ELISA results, and the 95th percentile of the S/P ratios in 2015-2016, as explanatory variables. As internal validation, both models were applied to predict positive ELISA results from January 2019-June 2021, in 8026 herds with Status A in January 2019. The model predicting ≥1 positive sample had an area under the receiver operating characteristics curve of 0.76 (95% CI: 0.75, 0.77). At a cut-off predicted probability πc = 0.40, 25% of Status A herds would be alerted with positive and negative predictive values of 0.52 and 0.83, respectively. The model predicting ≥2 positive samples had lower positive, but higher negative, predictive values. This study indicates that discrimination of Status A herds with high and low risks of future positive ELISA results is feasible. This might stimulate farmers with the highest risks to take additional measures to control any undetected Map infections.

Dairy Sheep Played a Minor Role in the 2005-2010 Human Q Fever Outbreak in The Netherlands Compared to Dairy Goats.

Q fever is an almost ubiquitous zoonosis caused by Coxiella burnetii. This organism infects several animal species, as well as humans, and domestic ruminants like cattle, sheep and goats are an important animal reservoir of C. burnetii. In 2007, a sudden rise in notified human Q fever cases occurred in The Netherlands, and by the end of 2009, more than 3500 human Q fever patients had been notified. Dairy sheep and dairy goats were suspected to play a causal role in this human Q fever outbreak, and several measures were taken, aiming at a reduction of C. burnetii shedding by infected small ruminants, in order to reduce environmental contamination and thus human exposure. One of the first measures was compulsory notification of more than five percent abortion within thirty days for dairy sheep and dairy goat farms, starting 12 June 2008. After notification, an official farm inspection took place, and laboratory investigations were performed aiming at ruling out or demonstrating a causal role of C. burnetii. These measures were effective, and the number of human Q fever cases decreased; levels are currently the same as they were prior to 2007. The effect of these measures was monitored using a bulk tank milk (BTM) PCR and an antibody ELISA. The percentage PCR positive dairy herds and flocks decreased over time, and dairy sheep flocks tested PCR positive significantly less often and became PCR negative earlier compared to dairy goat herds. Although there was no difference in the percentage of dairy goat and dairy sheep farms with a C. burnetii abortion outbreak, the total number of shedding dairy sheep was much lower than the number of shedding dairy goats. Combined with the fact that Q fever patients lived mainly in the proximity of infected dairy goat farms and that no Q fever patients could be linked directly to dairy sheep farms, although this may have happened in individual cases, we conclude that dairy sheep did not play a major role in the Dutch Q fever outbreak. BTM monitoring using both a PCR and an ELISA is essential to determine a potential C. burnetii risk, not only for The Netherlands but for other countries with small ruminant dairy industries.

Mycobacterium avium subsp. paratuberculosis ELISA Responses in Milk Samples from Vaccinated and Nonvaccinated Dairy Goat Herds in The Netherlands.

The aims of our study were to calculate the most appropriate cut-off value for milk samples in a serum-validated Mycobacterium avium subsp. paratuberculosis (MAP) ELISA and to analyze MAP ELISA responses in milk samples from vaccinated and nonvaccinated dairy goats in the Netherlands. Analyzed herds were representative for location and herd size of dairy goat herds in the Netherlands. A significantly higher proportion of the analyzed 49 herds were organic as compared with the total Dutch dairy goat population. First, the MAP ELISA was optimized using 992 paired serum and milk samples. At a cut-off of 25 S/P%, the relative sensitivity (Se) was 58.4% (n = 992, 95% CI: 48.8%-67.6%) and relative specificity (Sp) was 98.5% (n = 992, 95% CI: 97.5%-99.2%), as compared to serum ELISA results. The percentage of positively tested herds was 78.2% (n = 49, 95% CI: 63.4%-88.1%). The percentage of positive milk samples per herd (n = 22) was on average 4.6% (median, min, and max of 4.7%, 0.0%, and 10.7%, respectively). Average age of ELISA-positive (3.2 years) and -negative goats (3.2 years) was not different. Significantly more vaccinated goats tested positive (6.7%) as compared with nonvaccinated goats (1.1%). This study shows that a high number of vaccinated and nonvaccinated commercial dairy goat herds in the Netherlands have MAP-ELISA-positive goats.

Prostasomes: extracellular vesicles from the prostate.

The term 'prostasomes' is generally used to classify the extracellular vesicles (EVs) released into prostatic fluid by prostate epithelial cells. However, other epithelia within the male reproductive tract also release EVs that mix with 'true' prostasomes during semen emission or ejaculation. Prostasomes have been proposed to regulate the timing of sperm cell capacitation and induction of the acrosome reaction, as well as to stimulate sperm motility where all three are prerequisite processes for spermatozoa to attain fertilising capacity. Other proposed functions of prostasomes include interfering with the destruction of spermatozoa by immune cells within the female reproductive tract. On the other hand, it is unclear whether the distinct presumed functions are performed collectively by a single type of prostasome or by separate distinct sub-populations of EVs. Moreover, the exact molecular mechanisms through which prostasomes exert their functions have not been fully resolved. Besides their physiological functions, prostasomes produced by prostate tumour cells have been suggested to support prostate cancer spread development, and prostasomes in peripheral blood plasma may prove to be valuable biomarkers for prostate cancer.

Distinct lipid compositions of two types of human prostasomes.

Prostasomes are vesicles secreted by prostate epithelial cells and found in abundance in seminal plasma. They regulate aspects of sperm cell function and are also thought to prevent immune-mediated destruction of sperm cells within the female reproductive tract. In a previous study, we isolated two distinct populations of prostasomes, differing both in size and protein composition, from the seminal fluid of vasectomized men. In the current study, we characterized the lipid content of these two prostasome populations. Both prostasome types had an unusual lipid composition, with high levels of sphingomyelin (SM), cholesterol, and glycosphingolipids at the expense of, in particular, phosphatidylcholine. The different classes of glycerophospholipids consisted mainly of mono-unsaturated species. The sphingosine-based lipids, SM and the hexosylceramides, were characterized by a near absence of unsaturated species. The two types of prostasome differed in lipid composition, particularly with regard to the relative contributions of SM and hexosylceramides. Potential implications of the lipid compositions of prostasomes for the mechanisms of their formation and function are discussed.

Spermatozoa recruit prostasomes in response to capacitation induction.

Seminal plasma contains various types of extracellular vesicles, including 'prostasomes'. Prostasomes are small vesicles secreted by prostatic epithelial cells that can be recruited by and fuse with sperm cells in response of progesterone that is released by oocyte surrounding cumulus cells. This delivers Ca(2+) signaling tools that allow the sperm cell to gain hypermotility and undergo the acrosome reaction. Conditions for binding of prostasomes to sperm cells are however unclear. We found that classically used prostasome markers are in fact heterogeneously expressed on distinct populations of small and large vesicles in seminal plasma. To study interactions between prostasomes and spermatozoa we used the stallion as a model organism. A homogeneous population of ~60nm prostasomes was first separated from larger vesicles and labeled with biotin. Binding of biotinylated prostasomes to individual live spermatozoa was then monitored by flow cytometry. Contrary to assumptions in the literature, we found that such highly purified prostasomes bound to live sperm only after capacitation had been initiated, and specifically at pH ≥7.5. Using fluorescence microscopy, we observed that prostasomes bound primarily to the head of live sperm. We propose that in vivo, prostasomes may bind to sperm cells in the uterus, to be carried in association with sperm cells into oviduct and to fuse with the sperm cell only during the final approach of the oocyte. This article is part of a Special Issue entitled: An Updated Secretome.

Identification of distinct populations of prostasomes that differentially express prostate stem cell antigen, annexin A1, and GLIPR2 in humans.

In addition to sperm cells, seminal fluid contains various small membranous vesicles. These include prostasomes, membrane vesicles secreted by prostate epithelial cells. Prostasomes have been proposed to perform a variety of functions, including modulation of (immune) cell activity within the female reproductive tract and stimulation of sperm motility and capacitation. How prostasomes mediate such diverse functions, however, remains unclear. In many studies, vesicles from the seminal plasma have been categorized collectively as a single population of prostasomes; in fact, they more likely represent a heterogeneous mixture of vesicles produced by different reproductive glands and secretory mechanisms. We here characterized membranous vesicles from seminal fluid obtained from vasectomized men, thereby excluding material from the testes or epididymides. Two distinct populations of vesicles with characteristic sizes (56 ± 13 nm vs. 105 ± 25 nm) but similar equilibrium buoyant density (∼1.15 g/ml) could be separated by using the distinct rates with which they floated into sucrose gradients. Both types of vesicle resembled exosomes in terms of their buoyant density, size, and the presence of the ubiquitous exosome marker CD9. The protein GLIPR2 was found to be specifically enriched in the lumen of the smaller vesicles, while annexin A1 was uniquely associated with the surface of the larger vesicles. Prostate stem-cell antigen (PSCA), a prostate-specific protein, was present on both populations, thereby confirming their origin. PSCA was, however, absent from membrane vesicles in the seminal fluid of some donors, indicating heterogeneity of prostasome characteristics between individuals.

Quantitative and qualitative flow cytometric analysis of nanosized cell-derived membrane vesicles.

Nanosized cell-derived membrane vesicles are increasingly recognized as therapeutic vehicles and high-potential biomarkers for several diseases. Currently available methods allow bulk analysis of vesicles but are not suited for accurate quantification and fail to reveal phenotypic heterogeneity in membrane vesicle populations. For such analyses, single vesicle-based, multiparameter, high-throughput methods are needed. We developed a fluorescence-based, high-resolution flow cytometric method for quantitative and qualitative analysis of nanosized membrane vesicles. Proof of principle was obtained by single-particle analysis of virions and liposomes. Further validation was obtained by quantification of cell-derived nanosized membrane vesicles from cell cultures and body fluids. An important aspect was that the technology was extended to detect specific proteins on individual vesicles. This allowed identification of exosome subsets and phenotyping of individual exosomes produced by dendritic cells (DCs) undergoing different modes of activation. The described technology allows quantitative, multiparameter, and high-throughput analysis of a wide variety of nanosized particles and has broad applications. FROM THE CLINICAL EDITOR: The authors developed a fluorescence-based, high-resolution flow cytometric method for quantitative and qualitative analysis of nanosized cell-derived membrane vesicles that are increasingly recognized both as therapeutic vehicles and high-potential biomarkers for several diseases. A high throughput, easily available, and sensitive detection method such as the one discussed here is a critically important prerequisite for further refinements of this technology.

MHC class II-associated proteins in B-cell exosomes and potential functional implications for exosome biogenesis.

Professional antigen-presenting cells secrete major histocompatibility complex class II (MHC II) carrying exosomes with unclear physiological function(s). Exosomes are first generated as the intraluminal vesicles (ILVs) of a specific type of multivesicular body, and are then secreted by fusion of this compartment with the plasma membrane. We have previously shown that in contrast to the sorting of MHC II at lysosomally targeted multivesicular bodies, sorting of MHC II into exosomes does not rely on MHC II ubiquitination. In search for proteins that drive the incorporation of MHC II into exosomes or functionally discriminate exosomal from plasma membrane MHC II, we first analyzed the total proteome of highly purified B cell-derived exosomes using sensitive and accurate mass spectrometry (MS), and identified 539 proteins, including known and not previously identified constituents. Using quantitative MS, we then identified a small subset of proteins that were specifically co-immunoprecipitated with MHC II from detergent-solubilized exosomes. These include HSC71, HSP90, 14-3-3ɛ, CD20 and pyruvate kinase type M2 (PKM2), and we speculate on the functionality of their interaction with exosomal MHC II.

High iNOS mRNA and protein levels during early third trimester suggest a role for NO in prelabor cervical ripening in the bovine.

Nitric oxide (NO) plays a key role in the processes leading to cervical softening prior to labor. Inducible nitric oxide synthase (iNOS) contributes most to the increased production of NO during labor, as demonstrated in the rat cervix, or at term pregnancy in women. Changes in expression of iNOS during late gestation have not yet been studied longitudinally in any species, because repeatedly taking biopsies could not be performed. iNOS mRNA (n = 6) and protein expression (n = 3) in serial cervical biopsies of pregnant pluriparous cows taken around days 225, 250, and 275 of pregnancy and within 1.5 hr after calving (d225, d250, d275 and parturition biopsies, respectively) were measured using quantitative RT-PCR and Western blotting. iNOS mRNA expression decreased from the d225 biopsy onwards, differences being significant between the d250 and d275 (P < 0.05) and between the d275 and parturition biopsies (P < 0.05). iNOS protein expression decreased from d225 to d250 onwards. Immunohistochemical analysis of biopsies showed, besides positive staining in endothelium and epithelium, which remained unchanged at different time points, that iNOS expressing cells in the connective tissue cells of early biopsies were predominantly spindle shaped (mostly smooth muscle cells and some fibroblasts). In the parturition biopsies, iNOS reactivity was mainly found in mononuclear leucocytes. These results lead us to suggest that iNOS from spindle shaped cells is involved in prepartum cervical ripening, while iNOS in mononuclear inflammatory cells may be important for local tissue repair mechanisms during postpartum cervical involution.