Bar adsorptive microextraction and liquid chromatography-diode array detection of synthetic cannabinoids in oral fluid.

In recent years, synthetic cannabinoids (SCs) have become a major public health issue. For this reason, there is a need for innovative analytical methods that allow its monitoring in biological matrices. In this work, we propose a novel methodology to screen eight SCs (AM-694, cumyl-5F-PINACA, MAM-2201, 5F-UR-144, JWH-018, JWH-122, UR-144 and APINACA) in oral fluids. A bar adsorptive microextraction method followed by microliquid desorption combined with high-performance liquid chromatography with diode array detection (BAµE-µLD/HPLC-DAD) was developed to monitor the target SCs. The main factors affecting the BAµE technology were fully optimized for oral fluid analysis. Under optimized experimental conditions, the proposed methodology showed good linear dynamic ranges from 20.0 to 2000.0 µg L-1 (r2 > 0.99, relative residuals < 15%), limits of detection between 2.0 and 5.0 µg L-1 and suitable average recovery yields (87.9-100.5%) for the eight studied SCs. The intra- and interday accuracies (bias ≤ ± 14.7%) and precisions (RSD ≤ 14.9%) were also evaluated at three spiking levels. The validated methodology was then assayed to oral fluid samples collected from several volunteers. The proposed analytical approach showed remarkable performance and could be an effective alternative for routine monitoring of the target compounds in oral fluid.

Label-Free Detection of African Swine Fever and Classical Swine Fever in the Point-of-Care Setting Using Photonic Integrated Circuits Integrated in a Microfluidic Device.

Swine viral diseases have the capacity to cause significant losses and affect the sector's sustainability, a situation further exacerbated by the lack of antiviral drugs and the limited availability of effective vaccines. In this context, a novel point-of-care (POC) diagnostic device incorporating photonic integrated circuits (PICs), microfluidics and information, and communication technology into a single platform was developed for the field diagnosis of African swine fever (ASF) and classical swine fever (CSF). The device targets viral particles and has been validated using oral fluid and serum samples. Sensitivity, specificity, accuracy, precision, positive likelihood ratio (PLR), negative likelihood ratio (NLR), and diagnostic odds ratio (DOR) were calculated to assess the performance of the device, and PCR was the reference method employed. Its sensitivities were 80.97% and 79%, specificities were 88.46% and 79.07%, and DOR values were 32.25 and 14.21 for ASF and CSF, respectively. The proposed POC device and PIC sensors can be employed for the pen-side detection of ASF and CSF, thus introducing novel technological advancements in the field of animal diagnostics. The need for proper validation studies of POC devices is highlighted to optimize animal biosecurity.

The use of oral fluids and sock samples for monitoring key pathogens in pig populations for surveillance purposes.

Despite the prevalence of co-infections and the association of over 50 viral and 46 bacterial pathogens with pig diseases, little is known about their simultaneous occurrence, particularly in commercial pig farming environments where health programs are in place. To address this knowledge gap, this study aimed to evaluate the pathogen threshold of respiratory and enteric pathogens in pig herds using the Pork MultiPath™ (PMP1 and PMP2, respiratory and enteric respectively) technology, which detects multiple pathogens simultaneously in a single reaction with high sensitivity and specificity. In this study the most prevalent respiratory pathogens, Mycoplasma hyrohinis, Pasteurella multocida, and Haemophilus parasuis detected by PMP1 were effectively controlled during the nursery stage through strategic treatment with tiamulin. Even though the major respiratory incidences were reduced, the recorded coughing and sneezing rates were associated with the levels of H. parasuis and M. hyrohinis, which were set at 1356 and 1275 copies/reaction, respectively. In addition, one of the identified co-infection patterns indicated a strong relationship between the occurrence of H. parasuis and M. hyorhinis at the sample and pen levels, highlighting the high likelihood of detecting these two pathogens together. Testing with enteric panel PMP2 revealed that the most frequently detected virulence factors during the early nursery stage were Escherichia coli genes for toxins - ST1, ST2, and fimbriae - F4 and F18. Moreover, a co-infection with Rotavirus B and C was often observed during the nursery stage, and a strong positive correlation between these two markers has been identified. Additionally, the levels of several markers, namely E. coli F4, F5, F18, LT, ST1, and ST2, have been associated with a higher likelihood of sickness in pig populations. In addition, the onset of Brachyspira pilosicoli during the nursery and grower stages was found to be associated with an increased risk of diarrhoea, with a set threshold at around 500 copies/reaction. Although simultaneous detection of multiple pathogens is not yet widely used in the pig industry, it offers a significant advantage in capturing the diversity and interactions of co-infections. Testing pooled samples with Pork MultiPath™ is cost-effective and practical to regularly monitor the health status of pig populations.

Oral Fluids for the Early Detection of Classical Swine Fever in Commercial Level Pig Pens.

The early detection of classical swine fever (CSF) remains a key challenge, especially when outbreaks are caused by moderate and low-virulent CSF virus (CSFV) strains. Oral fluid is a reliable and cost-effective sample type that is regularly surveilled for endemic diseases in commercial pig herds in North America. Here, we explored the possibility of utilizing oral fluids for the early detection of CSFV incursions in commercial-size pig pens using two independent experiments. In the first experiment, a seeder pig infected with the moderately-virulent CSFV Pinillos strain was used, and in the second experiment, a seeder pig infected with the highly-virulent CSFV Koslov strain was used. Pen-based oral fluid samples were collected daily and individual samples (whole blood, swabs) every other day. All samples were tested by a CSFV-specific real-time RT-PCR assay. CSFV genomic material was detected in oral fluids on the seventh and fourth day post-introduction of the seeder pig into the pen, in the first and second experiments, respectively. In both experiments, oral fluids tested positive before the contact pigs developed viremia, and with no apparent sick pigs in the pen. These results indicate that pen-based oral fluids are a reliable and convenient sample type for the early detection of CSF, and therefore, can be used to supplement the ongoing CSF surveillance activities in North America.

Pen-Based Swine Oral Fluid Samples Contain Both Environmental and Pig-Derived Targets.

Laboratory methods for detecting specific pathogens in oral fluids are widely reported, but there is little research on the oral fluid sampling process itself. In this study, a fluorescent tracer (diluted red food coloring) was used to test the transfer of a target directly from pigs or indirectly from the environment to pen-based oral fluid samples. Pens of ~30, ~60, and ~125 14-week-old pigs (32 pens/size) on commercial swine farms received one of two treatments: (1) pig exposure, i.e., ~3.5 mL of tracer solution sprayed into the mouth of 10% of the pigs in the pen; (2) environmental exposure, i.e., 20 mL of tracer solution was poured on the floor in the center of the pen. Oral fluids collected one day prior to treatment (baseline fluorescence control) and immediately after treatment were tested for fluorescence. Data were evaluated by receiver operating characteristic (ROC) analysis, with Youden's J statistic used to set a threshold. Pretreatment oral fluid samples with fluorescence responses above the ROC threshold were removed from further analysis (7 of 96 samples). Based on the ROC analyses, oral fluid samples from 78 of 89 pens (87.6%), contained red food coloring, including 43 of 47 (91.5%) pens receiving pig exposure and 35 of 42 (83.3%) pens receiving environmental exposure. Thus, oral fluid samples contain both pig-derived and environmental targets. This methodology provides a safe and quantifiable method to evaluate oral fluid sampling vis-à-vis pen behavior, pen size, sampling protocol, and target distribution in the pen.

Dynamics of antibody response and bacterial shedding of Mycoplasma hyorhinis and M. hyosynoviae in oral fluids from experimentally inoculated pigs.

Mycoplasma hyorhinis (Mhr) and M. hyosynoviae (Mhs) are commensal organisms of the upper respiratory tract and tonsils but may also cause arthritis in pigs. In this study, 8-week-old cesarean-derived colostrum-deprived (CDCD) pigs (n = 30; 3 groups, 10 pigs per group, 2 pigs per pen) were inoculated with Mhr, Mhs, or mock-inoculated with culture medium and then pen-based oral fluids were collected at different time points over the 56 days of the experimental study. Oral fluids tested by Mhr and Mhs quantitative real-time PCRs revealed Mhr DNA between day post inoculation (DPI) 5-52 and Mhs DNA between DPI 5-15. Oral fluids were likewise tested for antibody using isotype-specific (IgG, IgA, IgM) indirect ELISAs based on a recombinant chimeric polypeptide of variable lipoproteins (A-G) for Mhr and Tween 20-extracted surface proteins for Mhs. Mhr IgA was detected at DPI 7 and, relative to the control group, significant (p < 0.05) antibody responses were detected in the Mhr group between DPI 12-15 for IgM and DPI 36-56 for both IgA and IgG. In the Mhs group, IgM was detected at DPI 10 and significant (p < 0.05) IgG and IgA responses were detected at DPI 32-56 and DPI 44-56, respectively. This study demonstrated that oral fluid could serve as an effective and convenient antemortem sample for monitoring Mhr and Mhs in swine populations.

Comparative analysis on efficiency and accuracy of parallel confocal microscopy and three-dimensional in motion video with triangulation technology-based intraoral scanner under influence of moisture and mouth opening - A crossover clinical trial.

Aim: The intraoral scanners are digital devices used to digitise the oral tissues. The accuracy of the intraoral scanners has been studied under different environmental conditions, but there might be differences that occur in the actual oral environment, which is still in question. The aim of the study was to evaluate the accuracy and efficiency of Parallel Confocal Microscopy and 3D in motion video with triangulation technology-based intraoral scanners under the influence of moisture and mouth opening. Settings and Design: This was an Cross over clinical controlled study. Materials and Methods: The controlled in vivo study included healthy subjects who were in need of CBCT for the purpose of locating the position of unerupted third molars before going abroad for a job. The subjects were exposed to scans in the upper and lower jaws with two intraoral scanners based on 3D motion video technology with triangulation (Medit) and parallel confocal microscopy (Trios) under the influence of two oral conditions, which were moisture (presence and absence of moisture) and mouth opening (30 mm and 50 mm, respectively). A total of 96 scans were obtained and superimposed individually over the reference CBCT scans to find the deviations in the Geomagic Rapidform (version 2020, USA) software. The efficiency of the scanners was calculated by recording the time taken and the number of images obtained after each scan. Statistical Analysis Used: The significance was calculated by using the independent and paired sample t test in SPSS software (IBM, version 23). Results: Based on the surface analysis, the trueness of the intra-oral scanners had statistically significant differences when compared between 3D in motion video technology with Triangulation and Parallel Confocal Microscopy (P < 0.05) whereas no statistical significance was observed in precision. There was a significant difference observed in the efficiency of the intra-oral scanners (P < 0.05). Conclusion: There is a significant difference in the accuracy and efficiency of the intraoral scanners under the influence of oral conditions, such as different moisture levels and mouth opening conditions. 3D in motion video technology with Triangulation showed better results with the least deviation than Parallel Confocal Microscopy.

Retrospective Analysis of the Detection of Pathogens Associated with the Porcine Respiratory Disease Complex in Routine Diagnostic Samples from Austrian Swine Stocks.

The diagnostic workup of respiratory disease in pigs is complex due to coinfections and non-infectious causes. The detection of pathogens associated with respiratory disease is a pivotal part of the diagnostic workup for respiratory disease. We aimed to report how frequently certain viruses and bacteria were detected in samples from pigs with respiratory symptoms in the course of routine diagnostic procedures. Altogether, 1975 routine diagnostic samples from pigs in Austrian swine stocks between 2016 and 2021 were analysed. PCR was performed to detect various pathogens, including porcine reproductive and respiratory syndrome virus (PRRSV) (n = 921), influenza A virus (n = 479), porcine circovirus type 2 (PCV2) (n = 518), Mycoplasma (M.) hyopneumoniae (n = 713), Actinobacillus pleuropneumoniae (n = 198), Glaesserella (G.) parasuis (n = 165) and M. hyorhinis (n = 180). M. hyorhinis (55.1%) had the highest detection rate, followed by PCV2 (38.0%) and Streptococcus (S.) suis (30.6%). PRRSV was detected most frequently in a pool of lung, tonsil and tracheobronchial lymph node (36.2%). G. parasuis was isolated more frequently from samples taken after euthanasia compared to field samples. PRRSV-positive samples were more likely to be positive for PCV2 (p = 0.001), M. hyopneumoniae (p = 0.032) and Pasteurella multocida (p < 0.001). M. hyopneumoniae-positive samples were more likely to be positive for P. multocida (p < 0.001) and S. suis (p = 0.046), but less likely for M. hyorhinis (p = 0.004). In conclusion, our data provide evidence that lung samples that were positive for a primary pathogenic agent were more likely to be positive for a secondary pathogenic agent.

Accuracy of periodontitis diagnosis obtained using multiple molecular biomarkers in oral fluids: A systematic review and meta-analysis.

AIM: To determine the accuracy of biomarker combinations in gingival crevicular fluid (GCF) and saliva through meta-analysis to diagnose periodontitis in systemically healthy subjects. METHODS: Studies on combining two or more biomarkers providing a binary classification table, sensitivity/specificity values or group sizes in subjects diagnosed with periodontitis were included. The search was performed in August 2022 through PUBMED, EMBASE, Cochrane, LILACS, SCOPUS and Web of Science. The methodological quality of the articles selected was evaluated using the QUADAS-2 checklist. Hierarchical summary receiver operating characteristic modelling was employed to perform the meta-analyses (CRD42020175021). RESULTS: Twenty-one combinations in GCF and 47 in saliva were evaluated. Meta-analyses were possible for six salivary combinations (median sensitivity/specificity values): IL-6 with MMP-8 (86.2%/80.5%); IL-1ß with IL-6 (83.0%/83.7%); IL-1ß with MMP-8 (82.7%/80.8%); MIP-1α with MMP-8 (71.0%/75.6%); IL-1ß, IL-6 and MMP-8 (81.8%/84.3%); and IL-1ß, IL-6, MIP-1α and MMP-8 (76.6%/79.7%). CONCLUSIONS: Two-biomarker combinations in oral fluids show high diagnostic accuracy for periodontitis, which is not substantially improved by incorporating more biomarkers. In saliva, the dual combinations of IL-1ß, IL-6 and MMP-8 have an excellent ability to detect periodontitis and a good capacity to detect non-periodontitis. Because of the limited number of biomarker combinations evaluated, further research is required to corroborate these observations.

Validity and reliability of in-person and remote oral fluids drug testing compared to urine drug testing.

BACKGROUND: Increased telehealth use has led to greater interest in remote drug testing. The speed, acceptability, and ability to observe oral fluids testing makes it the best candidate for remote drug testing, but its validity and reliability compared to gold-standard urine drug testing have not been established. METHODS: Veterans (N = 99) recruited from mental health clinics completed in-person and remote oral fluids testing and in-person urine drug testing. The validity of oral fluids versus urine drug testing and reliability of in-person versus remote oral fluids testing were evaluated. RESULTS: Validity of oral fluids testing was similar for samples collected in-person and virtually. Oral fluids testing had good specificity (0.93-1.00) and negative predictive value (0.85-1.00), but lower sensitivity and positive predictive value. Sensitivity (0.21-0.93) was highest for methadone and oxycodone, followed by cocaine and then amphetamine and opiates. Positive predictive value (0.14-1.00) was highest for cocaine, opiates, and methadone, followed by oxycodone and then amphetamine. Validity for cannabis was low, likely because of differences in detection windows for oral fluids versus urine drug screens. Reliability of remote oral fluids testing was adequate for opiates, cocaine, and methadone, but not oxycodone, amphetamine, or cannabis. CONCLUSIONS: Oral fluids testing identifies most negative, but not most positive, drug test results. While oral fluids testing is appropriate in some circumstances, its limitations should be acknowledged. Remote drug testing addresses many barriers, but also generates new barriers related to self-administration and remote interpretation. Limitations include a small sample and low base rates for some drugs.

Determination of synthetic cannabinoids in oral fluids by liquid chromatography with fluorescence detection after solid-phase extraction.

Synthetic cannabinoids are one of the most consumed new psychoactive substances, being absolutely necessary the development of analytical methodologies for the determination of these substances in biological fluids. In this study, a liquid chromatography with fluorescence detection (LC-FD) method has been developed for the analysis of 8 synthetic cannabinoids in oral fluids. The method has been validated in terms of linearity, precision and extraction recoveries, giving limits of detection as low as 0.7 µg L-1, and limits of quantification of 2.6 µg L-1. Different silica and polymeric commercial solid sorbents such as C18, Supel-Select HLB, EB2 ExtrabondⓇ and SampliQ-OPT were tested, concluding that Supel-Select HLB provided quantitative recoveries for the extraction of synthetic cannabinoids in oral fluids.•Analysis of synthetic cannabinoids in oral fluids.•Analytical procedure based on liquid chromatography with fluorescence detection.•Sample treatment based on solid phase extraction with HLB cartridges.

Determination of synthetic hallucinogens in oral fluids by microextraction by packed sorbent and liquid chromatography-tandem mass spectrometry.

A fast and simple procedure based on microextraction by packed sorbent (MEPS) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) has been developed for the simultaneous quantification of 28 synthetic hallucinogens in oral fluids, including lysergic acid diethylamide and substances from NBOMe, NBOH, NBF, 2C, and substituted amphetamine categories. Extraction conditions such as type of sorbent, sample pH, number of charge/discharge cycles, and elution volume were studied. Hallucinogenic compounds were extracted from oral fluid samples using C18 MEPS, loading with 100 µL sample (adjusted to pH 7) in 3 cycles, washing with 100 µL deionized water, and eluting with 50 µL methanol in 1 cycle, giving quantitative recoveries and no significant matrix effects. Limits of detection from 0.09 to 1.22 µg L-1; recoveries from 80 to 129% performed in spiked oral fluid samples at 20, 50, and 100 µg L-1; and high precision with relative standard deviations lower than 9% were obtained. The proposed methodology was demonstrated to be appropriate for the simple and sensitive determination of NBOMe derivates and other synthetic hallucinogenic substances in oral fluid samples.

Development of paper-immobilized molecularly imprinted polymers by laser pointer activation for methamphetamine extraction with analysis by ion mobility spectrometry.

A fast, simple, cheap, and versatile strategy has been proposed for the synthesis of paper-immobilized molecularly imprinted polymers (MIPs) by photoactivated bulk polymerization over a piece of nitrocellulose using a 405 nm laser pointer. Polymerization was carried out using a mixture of methacrylic acid and ethylene glycol dimethacrylate, using methamphetamine as template molecule and bis(2,4,6-trimethylbenzoyl)phenylphosphine oxide as radical initiator. After investigation of different polymerization parameters, the following experimental conditions were found to give best results: size of nitrocellulose strip (13.5 × 4.0 × 0.8 mm), type of porogen (acetonitrile), polymerization mixture volume (75 µL), and irradiation times (10 min). Experimental conditions (such as sample pH, extraction and desorption time, and type and volume of desorption solvent) were also adjusted for the extraction of methamphetamine using the proposed paper-MIP. Methamphetamine determination was carried out by ion mobility spectrometry providing a limit of detection of 14 µg L-1 and quantitative recoveries from 81 to 95% using spiked urine and oral fluid samples. The proposed paper-immobilized MIP device allows a simple and selective sample extraction procedure for the determination of methamphetamine in oral fluids and urine with a high portability, minimal solvent consumption, and reduced costs compared to other conventional approaches.

Randomly controlled drivers using minimally invasive sampling: assessment of drug prevalence in Western Switzerland over two time periods.

BACKGROUND: According to the World Health Organization, road traffic injuries lead to 1.3 million deaths each year and represent the leading cause of death for young adults under 30 years old. The use of psychoactive substances, including alcohol, drugs and pharmaceuticals, is a well-known risk factor for road traffic injuries. Our study aims to assess the prevalence of substances consumed by drivers in western Switzerland. Such studies are pivotal to improving prevention and developing public awareness campaigns. METHODS: To assess the prevalence of psychoactive substances among drivers, roadside controls were performed in collaboration with local police, using their classical sampling procedures to detect drivers under the influence of drugs or alcohol over two time periods (P1: 2006-2008, P2: 2017-2020). When impaired driving was not suspected by the police, minimally invasive sampling strategies (i.e., oral fluids during P1 and dried blood spots during P2) were performed on volunteer drivers after a road safety survey. A posteriori analyses and statistical interpretation were then performed. RESULTS: Among the 1605 drivers included in the study, 1048 volunteers provided an oral fluid sample, while 299 provided a dried blood spot sample. The percentage of drivers testing positive for at least one substance that can impact driving abilities was stable over time, with a rate of 10.5% positivity measured over both periods. Considering the different categories of substances, a slight variation was observed between both periods, with 7.6 and 6.3% of pharmaceuticals and 3.6 and 4.9% of illicit drugs for P1 and P2, respectively. Regarding the consumption of illicit drugs, the highest percentage of positivity was measured in biological fluids of drivers under the age of 35, during nights and week-ends, periods which are considered particularly prone to fatal accidents for this age group. Disturbingly, the road safety survey highlighted that drivers' perception of the risk of getting positively controlled while driving after drug consumption is low (3.3 on a 1-to-10 scale, N = 299). CONCLUSION: The number of positive cases measured in voluntary drivers who passed the preliminary police check demonstrates the importance of systematic biofluid sampling strategies regarding driving under the influence of psychoactive substances. Although the number of fatal road accidents globally has decreased over time, the results of this study reveal the need for both better prevention and deterrent processes that could potentially reduce the risk of fatal road accidents associated with drug consumption.

Porcine reproductive and respiratory syndrome virus RNA detection in tongue tips from dead animals.

The control of porcine reproductive and respiratory syndrome virus (PRRSV) hinges on monitoring and surveillance. The objective of this study was to assess PRRSV RNA detection by RT-PCR in tongue tips from dead suckling piglets compared to serum samples, processing fluids, and family oral fluids. Tongue tips and serum samples were collected from three PRRSV-positive breeding herd farms (farms A, B, and C) of three different age groups: newborns (<24 h), processing (2 to 7 days of age), and weaning (18 to 22 days of age). Additionally, processing fluids and family oral fluids were collected from 2-7 days of age and weaning age, respectively. In farms A and B, PRRSV RNA was detected in tongue tips from all age groups (100 and 95%, respectively). In addition, PRRSV RNA was detected in pooled serum samples (42 and 27%), processing fluids (100 and 50%), and family oral fluids (11 and 22%). Interestingly, the average Ct value from tongue tips was numerically lower than the average Ct value from serum samples in the newborn age. In farm C, PRRSV RNA was only detected in serum samples (60%) and family oral fluids (43%), both from the weaning age. Further, no PRRSV RNA was detected in tongue tips when pooled serum samples from the same age group tested PRRSV RNA-negative. Taken together, these results demonstrate the potential value of tongue tips for PRRSV monitoring and surveillance.

Effects of the Storage Conditions on the Stability of Natural and Synthetic Cannabis in Biological Matrices for Forensic Toxicology Analysis: An Update from the Literature.

The use and abuse of cannabis, be it for medicinal or recreational purposes, is widely spread among the population. Consequently, a market for more potent and consequently more toxic synthetic cannabinoids has flourished, and with it, the need for accurate testing of these substances in intoxicated people. In this regard, one of the critical factors in forensic toxicology is the stability of these drugs in different biological matrices due to different storage conditions. This review aims to present the most updated and relevant literature of studies performed on the effects of different storage conditions on the stability of cannabis compounds present in various biological matrices, such as blood and plasma, urine, and oral fluids, as well as in alternative matrices, such as breath, bile fluid, hair, sweat, cerumen, and dried blood spots.

Use of oral fluids for efficient monitoring of influenza viruses in swine herds in Colombia

Background: Influenza A virus (IAV) surveillance in swine is critical not only due to the direct impact of the disease in the pork industry but also because IAV are prone to interspecies transmission (from human to pigs and vice versa); therefore, its monitoring is fundamental from a public and animal health perspective. Several diagnostic techniques have been used to detect IAV infection from nasal samples in swine, while samples of oral fluids (OF) are in use as novel alternatives for pathogen detection. The OF allow for efficient and feasible low-cost disease detection at the herd level, with low risk of stress for the animals. Objective: To describe a surveillance strategy of IAV at the herd level during respiratory disease outbreaks in swine farms at tropical settings using porcine oral fluids. Methods: An active surveillance strategy was conducted in several farms with past records of respiratory disease. The IAV detection was conducted in five purposively selected swine farms from years 2014 to 2017. We investigated a total of 18 respiratory outbreaks of the disease. Swine OF were collected for IAV testing. An OF sample is described as a pen-based specimen collected from a group of >20 pigs per pen and/or per barn (stall-housed individually with close contact among them). The IAV infection was investigated in OF by rRT-PCR testing and confirmed by viral isolation in cell culture Results: We found 107 (7.4%) positives to IAV by rRT-PCR from a total of 1,444 OF samples tested. Additionally, 9 IAV isolates were all further identified as H1 subtype. Conclusions: Our results demonstrate that OF can be easily implemented as a novel, user-friendly, welfare-friendly, accurate and cost-effective sampling method for active surveillance and monitoring of IAV infections in swine farms in tropical settings.

Antecedentes: La vigilancia del Virus Influenza A (IAV) en los cerdos es fundamental debido al impacto directo de la enfermedad en la industria porcina, pero también porque los IAV son propensos de transmisión entre especies (humanos a cerdos y viceversa), y por lo tanto su monitoreo es crítico desde las perspectivas de salud pública y animal. Actualmente existen varias técnicas de diagnóstico disponibles para detectar la infección por IAV a partir de muestras nasales en cerdos, sin embargo, se han implementado otras muestras como los fluidos orales (OF) como nuevas alternativas para la detección de patógenos. El OF permite una detección eficiente y factible de enfermedades a menor costo a nivel de rebaño, con menor riesgo de estrés para los animales. Objetivo: Describir una estrategia de vigilancia de IAV a nivel de hato por medio de fluidos orales porcinos durante brotes de enfermedades respiratorias en granjas porcinas en entornos tropicales. Métodos: Se llevó a cabo una estrategia de vigilancia activa en cinco granjas porcinas seleccionadas con antecedentes de enfermedades respiratorias. Se recolectaron OF porcinos para la prueba de IAV. Una muestra de OF se describió como una muestra grupal recolectada de un grupo de >20 cerdos por corral y/o por establo (si estaban alojados individualmente, pero tenían un contacto cercano entre ellos). La infección por IAV se investigó probando OF mediante rRT-PCR y la confirmación mediante aislamiento viral en cultivo celular. Resultados: La detección de IAV se llevó a cabo en cinco granjas seleccionadas intencionalmente entre 2014- 2017. Investigamos un total de 18 eventos de brotes de enfermedades respiratorias. Del total de 1.444 OF muestras analizadas, encontramos 107 (7,4%) positivos a IAV mediante rRT-PCR. Además, solo se obtuvieron 9 aislamientos de IAV y todos se identificaron además como subtipo H1. Conclusiones: Los resultados de nuestro estudio demostraron cómo la OF puede implementarse fácilmente como un método de muestreo novedoso, fácil de usar, amigable con el bienestar animal, preciso y rentable para la vigilancia activa y el monitoreo de infecciones por IAV en granjas porcinas en entornos tropicales.

Antecedentes: A vigilância do vírus Influenza A (IAV) em suínos é crítica devido ao impacto direto da doença na indústria de suínos, mas também porque os IAV são propensos a transmissão interespécies (de humanos para porcos e vice-versa) e, portanto, seu monitoramento é crítico do ponto de vista da saúde pública e animal. Atualmente, existem várias técnicas de diagnóstico disponíveis para detectar a infecção por IAV em amostras nasais de suínos, no entanto, outras amostras, como fluidos orais (OF), têm sido implementadas como novas alternativas para a detecção de patógenos. O OF permite uma detecção eficiente e viável de doenças com menor custo em nível de rebanho, com menor risco de estresse para os animais. Objetivo: Descrever uma estratégia de vigilância de IAV em nível de rebanho durante surtos de doenças respiratórias em granjas de suínos em ambientes tropicais por meio de fluidos orais suínos. Métodos: A estratégia de vigilância ativa foi conduzida em cinco granjas de suínos selecionadas com histórico de doenças respiratórias. Suínos OF foram coletados para teste de IAV. Uma amostra OF foi descrita como um espécime baseado em curral coletado de um grupo de >20 porcos por curral e/ou por celeiro (se eles foram alojados individualmente, mas tendo contato próximo entre eles). A infecção IAV foi investigada testando OF por rRT-PCR e confirmada por isolamento em cultura de células. Resultados: A detecção do IAV foi realizada em cinco fazendas selecionadas propositalmente entre 2014-2017. Nós investigamos um total de 18 eventos de surto de doença respiratória. Do total de 1.444 amostras de OF testadas, encontramos 107 (7,4%) positivas para IAV por rRT-PCR. Além disso, apenas 9 isolados de IAV foram obtidos, e todos foram posteriormente identificados como subtipo H1. Conclusão: Os resultados de nosso estudo demonstraram como o OF pode ser facilmente implementado como um método de amostragem novo, amigável, amigável com o bem-estar, preciso e de baixo custo para vigilância ativa e monitoramento de infecções IAV em fazendas de suínos em ambientes tropicais.

Cross-Sectional Study on the Prevalence of PCV Types 2 and 3 DNA in Suckling Piglets Compared to Grow-Finish Pigs in Downstream Production.

Vertical transmission is a consistently discussed pathway of porcine circovirus type 2 (PCV2) and porcine circovirus type 3 (PCV3) transmission in pigs. To evaluate the presence of PCV2 and PCV3 in piglets, we collected tissue samples from 185 piglets that were crushed within the first week of life from 16 farms located in Germany and Austria. Pooled samples consisting of thymus, inguinal lymph node, myocardium, lung and spleen were examined for PCV2 and PCV3 by qPCR. Furthermore, oral fluid samples (OFS) from grow−finish pigs were collected and examined the same way. In piglets, PCV2 was highly prevalent (litters: 69.4%; piglets: 61.6%), whereas PCV3 prevalence was low (litters: 13.4%; piglets: 13.0%). In total, 72.6% and 67.2% of all collected OFS were PCV2 or PCV3 positive, respectively. Sow vaccination against PCV2 was identified as a protective factor concerning PCV2 in piglets (OR: 0.279; CI: 0.134−0.578; p < 0.001), whereas the porcine reproductive and respiratory syndrome virus (PRRSV) vaccination of sows was identified as a protective factor concerning PCV3 in piglets (OR: 0.252 CI: 0.104−0.610; p = 0.002). Our results show that PCV2, but not PCV3, is ubiquitous in suckling piglets and that early PCV3 infections might be modulated by PRRSV−PCV3 interaction. However, the ubiquitous nature of both viruses in older pigs could be confirmed.

Point-of-Care and Label-Free Detection of Porcine Reproductive and Respiratory Syndrome and Swine Influenza Viruses Using a Microfluidic Device with Photonic Integrated Circuits.

Swine viral diseases challenge the sector's sustainability by affecting productivity and the health and welfare of the animals. The lack of antiviral drugs and/or effective vaccines renders early and reliable diagnosis the basis of viral disease management, underlining the importance of point-of-care (POC) diagnostics. A novel POC diagnostic device utilizing photonic integrated circuits (PICs), microfluidics, and information and communication technologies for the detection of porcine reproductive and respiratory syndrome virus (PRRSV) and swine influenza A (SIV) was validated using spiked and clinical oral fluid samples. Metrics including sensitivity, specificity, accuracy, precision, positive likelihood ratio (PLR), negative likelihood ratio (NLR), and diagnostic odds ratio (DOR) were calculated to assess the performance of the device. For PRRSV, the device achieved a sensitivity of 83.5%, specificity of 77.8%, and DOR values of 17.66, whereas the values for SIV were 81.8%, 82.2%, and 20.81, respectively. The POC device and PICs can be used for the detection of PRRSV and SIV in the field, paving the way for the introduction of novel technologies in the field of animal POC diagnostics to further optimize livestock biosecurity.

Cough associated with the detection of Mycoplasma hyopneumoniae DNA in clinical and environmental specimens under controlled conditions.

BACKGROUND: The association of cough with Mycoplasma hyopneumoniae (MHP) DNA detection in specimens was evaluated under conditions in which the MHP status of inoculated and contact-infected pen mates was closely monitored for 59 days post-inoculation (DPI). METHODS: Seven-week-old pigs (n = 39) were allocated to five rooms (with one pen). Rooms contained 9 pigs each, with 1, 3, 6, or 9 MHP-inoculated pigs, respectively, except Room 5 (three sham-inoculated pigs). Cough data (2 × week) and specimens, tracheal swabs (2 × week), oral fluids (daily), drinker wipes (~ 1 × week), and air samples (3 × week) were collected. At 59 DPI, pigs were euthanized, and lung and trachea were evaluated for gross and microscopic lesions. Predictive cough value to MHP DNA detection in drinker and oral fluid samples were estimated using mixed logistic regression. RESULTS: Following inoculation, MHP DNA was first detected in tracheal swabs from inoculated pigs (DPI 3), then oral fluids (DPI 8), air samples (DPI 10), and drinker wipes (21 DPI). MHP DNA was detected in oral fluids in 17 of 59 (Room 1) to 43 of 59 (Room 3) samples, drinker wipes in 4 of 8 (Rooms 2 and 3) to 5 of 8 (Rooms 1 and 4) samples, and air samples in 5 of 26 (Room 2) or 3 of 26 (Room 4) samples. Logistic regression showed that the frequency of coughing pigs in a pen was associated with the probability of MHP DNA detection in oral fluids (P < 0.01) and nearly associated with drinker wipes (P = 0.08). Pathology data revealed an association between the period when infection was first detected and the severity of gross lung lesions. CONCLUSIONS: Dry, non-productive coughs suggest the presence of MHP, but laboratory testing and MHP DNA detection is required for confirmation. Based on the data from this study, oral fluids and drinker wipes may provide a convenient alternative for MHP DNA detection at the pen level when cough is present. This information may help practitioners in specimen selection for MHP surveillance.