Localization and distribution of goose astrovirus 2 antigens in different tissues at different times.

Goose astrovirus 2 (GAstV-2) causes visceral gout in goslings and has resulted in significant economic losses in the goose industry of China since its outbreak in 2017. To further investigate the distribution and localization of GAstV-2 in different tissues at different times, a monoclonal antibody (mAb)-based immunohistochemical (IHC) assay was developed to detect GAstV-2. A total of 80 1-day-old healthy goslings were inoculated with GAstV-2 via the oral (n = 40) and intramuscular routes (n = 40). GAstV-2 in the tissues of interest was detected using the established IHC assay. The results showed that positive signals were detected in most tissues at 1 day post-infection (dpi). Viral antigens were mainly distributed in the cytoplasm, and the staining intensity was higher in the renal tubular epithelial cells than in other cells. Taken together, our data demonstrated that GAstV-2 has a broad tissue tropism and primarily targets the kidneys. These results are likely to provide a scientific basis for further elucidation of the pathogenesis of GAstV-2.

Development of a droplet digital PCR method for detection of porcine circovirus 4.

BACKGROUND: Porcine circovirus 4 (PCV4), a newly emerging virus that was first discovered in 2019, may pose a potential threat to the pig industry. Droplet digital PCR (ddPCR) is an absolute quantitative method that has high sensitivity and accuracy. In this study, we developed a novel ddPCR assay to detect PCV4. Furthermore, we evaluated the detection limit, sensitivity, specificity and reproducibility of the ddPCR and TaqMan real-time quantitative PCR (qPCR) and tested 160 clinical samples to compare the detection rate of the two methods. RESULTS: The detection limit for ddPCR was 0.54 copies/µL, 10.6 times greater sensitivity than qPCR. Both ddPCR and qPCR assays exhibited good linearity and repeatability, and the established ddPCR method was highly specific for PCV4. The results of clinical sample testing showed that the positivity rate of ddPCR (5.6%) was higher than that of qPCR (4.4%). CONCLUSIONS: This study successfully developed a sensitive, specific and repeatable ddPCR assay for PCV4 detection, which can be widely used in clinical diagnosis of PCV4 infections.

Comparison of methods for determining Batrachochytrium dendrobatidis zoospore viability.

The emerging fungal pathogen Batrachochytrium dendrobatidis (Bd) threatens hundreds of amphibian species globally. During laboratory-based experiments it is often essential to quantify live Bd cells, but a comparison of the effectiveness of methods for counting and assessing the viability of the infectious zoospore life stage has not been done. A direct comparison of staining methods that assess viability will ensure that the most accurate and efficient method is used. Here, we compared the use of 2 relatively cheap common stains, trypan blue and methylene blue, and assessed their accuracy and precision for estimating the viability of Bd zoospores during both manual counting and colorimetric assays. We stained known proportions of killed Bd zoospores (0, 0.25, 0.50, 0.75, and 1.00) with each stain and estimated the proportion of stained (dead) and unstained (viable) cells in each sample using both manual counting and colorimetric assays. Trypan blue was found to be a much more effective stain than methylene blue for both microscopy and colorimetric assays. Additionally, counting zoospores via microscopy was both a more accurate and precise technique. We recommend using manual counts via microscopy using the trypan blue stain for assessing Bd zoospore viability.

Virulence and genetic differences among white spot syndrome virus isolates inoculated in Penaeus vannamei.

White spot syndrome virus (WSSV) infects several economically important aquaculture species, and has caused significant losses to the industry. This virus belongs to the Nimaviridae family and has a dsDNA genome ranging between 257 and 309 kb (more than 20 isolate genomes have been fully sequenced and published to date). Multiple routes of infection could be the cause of the high virulence and mortality rates detected in shrimp species. Particularly in Penaeus vannamei, differences in isolate virulence have been observed, along with controversy over whether deletions or insertions are associated with virulence gain or loss. The pathogenicity of 3 isolates from 3 localities in Mexico (2 from Sinaloa: 'CIAD' and 'Angostura'; and one from Sonora: 'Sonora') was evaluated in vivo in whiteleg shrimp P. vannamei infection assays. Differences were observed in shrimp mortality rates among the 3 isolates, of which Sonora was the most virulent. Subsequently, the complete genomes of the Sonora and Angostura isolates were sequenced in depth from infected shrimp tissues and assembled in reference to the genome of isolate strain CN01 (KT995472), comprising 289350 and 288995 bp, respectively. Three deletion zones were identified compared to CN01, comprising 15 genes, including 3 envelope proteins (VP41A, VP52A and VP41B), 1 non-structural protein (ICP35) and 11 other encoding proteins whose function is currently unknown. In addition, 5 genes (wsv129, wsv178, wsv204, wsv249 and wsv497) presented differences in their repetitive motifs, which could potentially be involved in the regulation of gene expression, causing virulence variations.

Keeping up with advances in qPCR pathogen detection: an example for QPX disease in hard clams.

With marine diseases on the rise and increased reliance on molecular tools for disease surveillance, validated pathogen detection capabilities are important for effective management, mitigation, and response to disease outbreaks. At the same time, in an era of continual evolution and advancement of molecular tools for pathogen detection, it is critical to regularly reassess previously established assays to incorporate improvements of common practices and procedures, such as the minimum information for publication of quantitative real-time PCR experiments (MIQE) guidelines. Here, we reassessed, re-optimized, and improved the quantitative PCR (qPCR) assay routinely used for Quahog Parasite Unknown (QPX) disease monitoring. We made 19 significant changes to the qPCR assay, including improvements to PCR amplification efficiency, DNA extraction efficiency, inhibition testing, incorporation of linearized standards for absolute quantification, an inter-plate calibration technique, and improved conversion from copy number to number of cells. These changes made the assay a more effective and efficient tool for disease monitoring and pathogen detection, with an improved linear relationship with histopathology compared to the previous version of the assay. To support the wide adoption of validated qPCR assays for marine pathogens, we provide a simple workflow that can be applied to the development of new assays, re-optimization of old or suboptimal assays, or assay validation after changes to the protocol and a MIQE-compliant checklist that should accompany any published qPCR diagnostic assay to increase experimental transparency and reproducibility amongst laboratories.

An exploration of angiotensin-converting enzyme (ACE) inhibitory peptides derived from gastrointestinal protease hydrolysate of milk using a modified bioassay-guided fractionation approach coupled with in silico analysis.

An improved bioassay-guided fractionation was performed to effectively screen angiotensin-I converting enzyme inhibitory (ACEI) peptides from milk protein hydrolysate. The aqueous normal phase liquid chromatography, namely hydrophilic interaction liquid chromatography (HILIC), was used as a format of solid-phase extraction (SPE) short column for the first fractionation, then the HILIC-SPE fraction with the best ACEI activity (IC50 = 61.75 ± 5.74 µg/mL; IC50 = half-maximal inhibitory concentration) was obtained when eluted by 95% acetonitrile + 0.1% formic acid (fraction F1). The best HILIC-SPE fraction was further fractionated using reversed-phase (RP)-SPE short column. The best RP-SPE fraction was obtained when eluted by 20% acetonitrile + 0.1% formic acid (fraction P3) with an ACEI activity of IC50 36.22 ± 1.18 µg/mL. After the 2-step fractionation, the IC50 value of fraction P3 significantly decreased by 8.92-fold when compared with the crude hydrolysate. Several peptides were identified from fraction P3 using liquid chromatography-tandem mass spectrometry. The in silico analysis of these identified sequences based on the BIOPEP database predicted that HLPLPLL (HL-7) was the most active peptide against angiotensin-converting enzyme (ACE). The HL-7 derived from ß-casein showed a potent ACEI activity (IC50 value is 16.87 ± 0.3 µM). The contents of HL-7 in the gastrointestinal protease hydrolysate and RP-SPE fraction originated from 1 mg of milk proteins were quantified using a multiple reaction monitoring mode upon liquid chromatography-tandem mass spectrometry analysis to give 19.86 ± 1.14 pg and 14,545.8 ± 572.9 pg, respectively. Besides, the kinetic study indicated that HL-7 was a competitive inhibitor and the result was rationalized using the docking simulation. The study demonstrated an efficient screening of ACEI peptides from commercially available milk powders using a simple SPE process instead of a sophisticated instrument such as HPLC. Moreover, the potent ACEI peptide HL-7 uncovered by this method could be a natural ACE inhibitor.

Effect of assay choice, viral concentration and operator interpretation on infectious bronchitis virus detection and characterization.

Despite the efforts to achieve a consistent classification scheme based on the complete S1 gene, the genetic characterization of infectious bronchitis virus (IBV) is often performed on partial S1 regions due to economic and time constraints in the diagnostic routine. Sanger sequencing remains the most common and cost-effective option even if the analysis of samples where multiple field and vaccine strain populations coexist can lead to partial or misleading results. The present study aimed to evaluate the different diagnostic outcomes of three commonly used RT-PCR methods targeting two regions of the S1 gene. A possible bias in IBV detection and characterization was investigated in relation to the adopted method, the strain concentration as well as their ratio in mixed samples. Thirty samples were prepared by artificially mixing two vaccine strains, combined at different ratios and selected among four different IBV lineages, i.e. GI-1 (Mass), GI-13 (793/B), GI-19 (QX), GI-23 (Israeli Variant 2). Sequence analysis was conducted both manually and with bioinformatic methods. The result agreement among methods, replicates and analysis approaches was statistically evaluated. Consistent results emerged among the three assays, with a few discrepancies likely caused by primer affinity and target amount. This study confirms the complexity of IBV strain identification and highlights the importance of evaluating and updating the available diagnostic assays for a reliable detection of all circulating IBV strains.

Development and evaluation of recombinase polymerase amplification combined with lateral flow dipstick assays for co-detection of epizootic haemorrhagic disease virus and the Palyam serogroup virus.

BACKGROUND: Epizootic haemorrhagic disease virus (EHDV) and the Palyam serogroup viruses (PALV) have led to significant economic losses associated with livestock production globally. A rapid, sensitive and specific method for the detection of EHDV and PALV is critical for virus detection, monitoring, and successful control and elimination of related diseases. RESULTS: In the present study, a recombinase polymerase amplification combined with lateral flow dipstick (RPA-LFD) assay for the co-detection of genome segment 1 (Seg-1) of EHDV and PALV was developed and evaluated. The analytical sensitivities of the established RPA-LFD assay in the detection of EHDV and PALV were 7.1 copies/µL and 6.8 copies/µL, respectively. No cross-reaction with other members of the genus Orbivirus, including African horse sickness virus, bluetongue virus, Guangxi orbivirus, Tibet orbivirus and Yunnan orbivirus was observed. The established RPA-LFD assay accurately detected 39 EHDV strains belonging to 5 serotypes and 29 PALV strains belonging to 3 serotypes. The trace back results of quantitative real-time polymerase chain reaction (qRT-PCR) and the established RPA-LFD assay on sentinel cattle were consistent. The coincidence rates of qRT-PCR and the established RPA-LFD assay in 56 blood samples from which EHDV or PALV had been isolated and 96 blood samples collected from cattle farms were more than 94.8 %. The results demonstrated that the established RPR-LFD assay is specific, sensitive and reliable, and could be applied in early clinical diagnosis of EHDV and PALV. CONCLUSIONS: This study highlights the development and application of the RPA-LFD assay in the co-detection of EHDV and PALV for the first time. The assay could be used as a potential optional rapid, reliable, sensitive and low-cost method for field diagnosis of EHDV and PALV.

Development of a molecular biological assay for the detection of markers related to decreased susceptibility to macrolides and lincomycin in Mycoplasma hyorhinis.

The control of Mycoplasma hyorhinis infection relies mainly on antimicrobial therapy. However, the antibiotic susceptibility testing of the bacteria is usually not performed before applying the treatment, and thus therapeutic failures are not uncommon. In the case of M. hyorhinis, several antibiotic-resistance-related single nucleotide polymorphisms (SNPs) are known but assays for their detection have not been described yet. The aims of the present study were to investigate macrolide- and lincomycin-resistance-related SNPs in Hungarian M. hyorhinis isolates and to develop mismatch amplification mutation assays (MAMA) to detect the identified resistance markers. Minimal inhibitory concentrations (MIC) of different drugs and whole genome sequences of 37 M. hyorhinis isolates were used to find the resistance-related mutations. One MAMA assay was designed to detect the mutation of the 23S rRNA gene at nucleotide position 2058 (Escherichia coli numbering). For further evaluation, the assay was challenged with 17 additional isolates with available MIC data and 15 DNA samples from clinical specimens. The genotypes of the samples were in line with the MIC test results. The developed assay supports the practice of targeted antibiotic usage; hence it may indirectly reduce some bacterial resistance-related public health concerns.

In vitro acaricidal activity of Cymbopogon citratus, Cymbopogon nardus and Mentha arvensis against Rhipicephalus microplus (Acari: Ixodidae).

The chemical composition and acaricidal activity of plant-derived essential oils was assessed against Rhipicephalus microplus ticks. The essential oils of Mentha arvensis, Cymbopogon citratus and C. nardus were assessed for acaricidal activity against Rhipicephalus microplus. Essential oils (EO) of plants were separated by hydrodistillation (three times) and analyzed using gas chromatography - mass spectrometer (GC-MS). For bioassays, engorged females of R. microplus were exposed to C. citratus and C. nardus EO at 2%, 3%, 4% and 5% concentrations; and to M. arvensis EO at 1%, 3%, and 5% for 5 min. The weight egg mass, nutrient index (N.I), egg production index (E.P.I), hatching and control rate were evaluated. Non-feed larvae of R. microplus were exposed to essential oils with 0.25%, 0.5%; 1%; 1.5% and 2% concentrations; the mortality rate was measured after 48 h. Only engorged females presented reduced biological activities (oviposition, E.P.I) after exposure to M. arvensis at 3%, when in comparison to both positive and negative controls. The hatchability of R. microplus larvae ranged from 66.9% (after exposure to C. nardus EO at 5%) to 99.2% (positive control). The nutrition index was lower (46.6%) for the exposure to M. arvensis EO at 5%. M. arvensis at 3% and 5% concentrations was significantly efficient for engorged females when compared to control (53.7% and 47.5%, respectively). C. citratus EO at 1%, 1.5% and 2% concentrations yielded better results in the larval packet test, causing 100% mortality. Nonetheless, C. nardus and M. arvensis EO at 2% yielded 66% and 39% mortality, respectively. The study showed that M. arvensis presented potential for the control of R. microplus engorged females while C. citratus and C. nardus presented potential as a larvicide.

Rhipicephalus microplus (acari: Ixodidae): Clinical safety and potential control by topical application of cottonseed oil (Gossypium sp.) on cattle.

The present study was performed to determine the acaricidal activity of the cottonseed oil (CSO) against cattle tick Rhipicephalus microplus. CSO was analyzed using Gas Chromatograph with high-resolution Mass Spectrometer (GC-HRMS) to identify the presence of active compounds. In vitro bioassays were performed using larval packet test (LPT) and adult immersion test (AIT) by taking different concentrations of CSO (i.e. 0.1, 0.5, 1.5, 2.5, 5, 7.5, 10 and 12.5%). In vivo acaricidal activity of CSO was evaluated by its topical application on red Sahiwal calves for 144 h. Clinical safety of CSO was evaluated by performing skin irritancy test and examination of hematological profile of calves'. GC-HRMS analysis of CSO revealed the presence of many fatty acids including oleic acid, lauric acid, palmitic acid, stearic acid and other components. Results exhibited that all the concentrations of CSO were effective in reducing the number of ticks and their growth. However, CSO at concentrations of 10% (CSO7) and 12.5% (CSO8) exhibited 100% mortality of R. microplus larvae and adults in LPT and AIT, respectively. In vivo acaricidal assay revealed that CSO7 and CSO8 shown 85% and 89% inhibition of ticks, respectively on calves after 144 h as compared to the control group. CSO was clinically safe on calves' skin with mild erythema up to 20 min. Hematological profile of calves revealed no sign of toxicity after treatment with CSO. Thus, CSO can be used as an alternative and safe drug therapy against R. microplus.

Experimental inoculation of Neospora caninum tachyzoites in eared doves (Zenaida auriculata).

Neospora caninum is an apicomplexan parasite distributed worldwide. Although a positive association between the presence of birds and abortions in cattle associated to N. caninum has been reported, the role of the birds in the epidemiologic cycle of the parasite is unknown. To the best knowledge, no experimental studies have evaluated N. caninum in the eared dove, Zenaida auriculata. Therefore, we aimed to determine whether Z. auriculat can act as intermediate host for N. caninum. Eighteen birds were divided into four groups, G1, G2, G3, and G4 (control); G1, G2 and G3 received 2 × 106 tachyzoites of NC-1 strain via different routes: subcutaneous, intramuscular, and intraperitoneal, respectively. G4 composed of three birds. Serum samples were collected weekly, and one bird each from G1, G2 and G3 was euthanized on the 7th and 14th day post-inoculation (dpi). The remaining birds were euthanized after the 28th dpi. Tissues from the doves were evaluated using histopathological analysis, PCR and dog bioassay to detect the parasite. Dogs were fed with tissues from the birds and monitored for 30 days. Serum samples were collected weekly from the dogs for serological analysis, and feces samples were collected daily until the end of the experiment for coproparasitological examinations. No dove showed clinical signs of the infection; however, all of them seroconverted after the inoculation, with stronger immunological response in the G3 birds. The lung tissue of one G3 bird showed positive PCR results; it was euthanized on the 7th dpi, and an inflammatory infiltrate was observed in the lung and kidney from this dove. The dogs did not shed oocysts or seroconverted. Our results indicate that the intraperitoneal route induced infection in the doves; however, the parasite may have been eliminated by the host, and the doves may be resistant to chronic infection.

Effect of entomopathogenic native fungi from paddock soils against Rhipicephalus microplus larvae with different toxicological behaviors to acaricides.

The objectives of this study are to: (1) evaluate the in vitro acaricidal effect of 54 Metarhizium anisopliae strains, six Beauveria bassiana strains and one Purpureocilium lilacinum strain, against the larvae of two populations of Rhipicephalus microplus (multi-resistant and susceptible to chemical acaricides); and (2) determine the lethal concentrations required to eliminate the 50% (LC50) and 99% (LC99) of larvae through the use of entomopathogenic fungi (EF) with high acaricidal effects. The mortality percentage was evaluated by larval immersion tests at a dose of 1 × 108 conidia/mL for each fungal strain. For calculating LC50 and LC99, four doses (1 × 108, 1 × 107, 1 × 106 and 1 × 105) were used. Nine strains of M. anisopliae and the P. lilacinum strain showed a high mortality percentage in the R. microplus larvae of both populations. The best strains that showed the lowest values of LC50 and LC99 for tick elimination were MaV50 and PlV01. In conclusion, several strains of entomopathogenic fungi showed a high acaricidal effect against the R. microplus larvae of both populations, suggesting that these fungi might be a promissory adjuvant in the control of R. microplus, including those who are resistant. Finally, the discovery of a P. lilacinum strain with a high acaricidal effect is also reported.

The pathogenicity of two Neospora caninum goat strains in a BALB/c mouse model.

Goats are frequently described as an intermediate host for the protozoan Neospora caninum, manifesting the disease mainly by recurrent abortions with placentitis and encephalitis in fetuses. Several reports of natural and experimental infections in cattle and mice show differences in the immune response, and the outcome of the infection can be variable depending on the species affected and by the behavior of the infective strain. This study describes for the first time two Neospora caninum strains isolated from naturally infected goats from the state of Minas Gerais, Brazil. One placenta and one brain from different goats were processed for a first bioassay in gerbils (Meriones unguiculatus). Subsequently, a second bioassay was performed by inoculating the processed brain samples from gerbils into Interferon gamma (IFN-γ) knockout mice (KO mice). Tachyzoites collected from the peritoneal fluid of the KO mice were inoculated into VERO cell monolayers, where they presented a very slow growth rate. The tachyzoites were also inoculated into BALB/c mice with a dose of 106 tachyzoites per animal. After a 5-week follow up, the animals infected with both of the strains developed a strong polarized Th1 response with increased serum and spleen gene expression levels of pro-inflammatory cytokines (mainly IFN-γ and TNF-α) in the first week. Tissue lesions were mild in the animals infected with both strains. Despite the strong immune response preventing an infection in the visceral organs, the parasite was able to reach the brain, causing progressive brain lesions from the second to fifth week post infection. The NC-goat1-infected mice presented with severe meningoencephalitis, but the NC-goat2-infected animals had considerable histological brain lesions only at week 5. Immunohistochemical analysis of the mouse brains revealed a different pattern of inflammatory cells compared to the naturally infected goats. A severe inflammatory infiltrate of CD3+ T lymphocytes was found in the NC-goat1-infected mice. A more discrete infiltrate of CD3+ T cells was found in the NC-goat2-infected animals. Additionally, IBA1 IHC revealed an intense microglial reaction and monocyte perivascular cuffs in the NC-goat1-infected animals and lower microglia/monocyte infiltrates in the NC-goat2-infected mice. This work contributes knowledge on the pathogenicity of new Neospora caninum strains in mice, comparable with other well-established mouse models of the disease, and demonstrates the importance of studying goats as an intermediate host of this parasite.

First description of clonal lineage type II (genotype #1) of Toxoplasma gondii in abortion outbreak in goats.

The purpose of this study was to perform genotypic characterization and to evaluate the virulence of Toxoplasma gondii obtained from aborted fetuses in an abortion outbreak in goats from northeastern Brazil. Brain samples from 32 fetuses were submitted to mouse bioassay for T. gondii isolation. Two isolates were obtained and subjected to genotypic characterization. Isolate virulence was evaluated using murine model in different doses (from 105 to 101 tachyzoites/mL). In genotyping, both isolates were classified as clonal lineage type II (genotype #1 ToxoDB) and showed to be virulent for mice. This is the first description of genotype #1 in cases of goat abortion, showing the circulation of virulent T. gondii isolate producing reproductive disorders in pregnant goat.

Analytical and diagnostic performance of a qPCR assay for Ichthyophonus spp. compared to the tissue culture 'gold standard'.

Parasites of the genus Ichthyophonus infect many fish species and have a non-uniform distribution within host tissues. Due in part to this uneven distribution, the comparative sensitivity and accuracy of using molecular-based detection methods versus culture to estimate parasite prevalence is under debate. We evaluated the analytical and diagnostic performance of an existing qPCR assay in comparison to the 'gold standard' culture method using Pacific herring Clupea pallasii with known exposure history. We determined that the assay is suitable for use in this host, and diagnostic specificity was consistently high (>98%) in both heart and liver tissues. Diagnostic sensitivity could not be fully assessed due to low infection rates, but our results suggest that qPCR is not as sensitive as culture under all circumstances. Diagnostic sensitivity of qPCR relative to culture is likely affected by the amount of sample processed. The prevalence values estimated by the 2 methods were not significantly different when sample amounts were equal (heart tissue), but when the assayed sample amounts were unequal (liver tissue), the culture method detected a significantly higher prevalence of the parasite than qPCR. Further, culture of liver also detected significantly more Ichthyophonus infections than culture of heart, suggesting that the density and distribution of parasites in tissues also plays a role in assay sensitivity. This sensitivity issue would be most problematic for fish with light infections. Although qPCR does not detect the presence of a live organism, DNA-based pathogen detection methods provide the opportunity for alternate testing strategies when culture is not possible.

Isolation and biological and molecular characterization of Neospora caninum (NC-SP1) from a naturally infected adult asymptomatic cattle (Bos taurus) in the state of São Paulo, Brazil.

The biological and genetic diversity of Neospora caninum is very limited because of availability of only a few viable isolates worldwide. This study describes the isolation and biological and molecular characterization of a new viable isolate of N. caninum (NC-SP1), from a cattle in Brazil. Approximately 400 g of brain from a naturally infected adult male cattle from an abattoir was fed to a 2-month-old dog. Neospora-like oocysts were observed on day 7 post-inoculation (PI) and the duration of oocyst shedding was 14 days. The DNA obtained from oocysts was characterized molecularly and the final sequence was 99% identical to homologous sequences of N. caninum available in GenBank®. For bioassay, gerbils (Meriones unguiculatus) were orally inoculated with 10 100 and 1000 oocysts; all gerbils remained clinically normal but developed N. caninum antibodies 14 days PI. Cell culture isolation was successful using the brain homogenate from one of the gerbils and tachyzoites were observed 24 days PI. Microsatellite genotyping revealed a unique genetic profile for this new reference isolate.

Evaluation of an accelerated hydrogen peroxide disinfectant to inactivate porcine epidemic diarrhea virus in swine feces on aluminum surfaces under freezing conditions.

BACKGROUND: Since its emergence in 2013, porcine epidemic diarrhea virus (PEDV) spread rapidly throughout the country due, in part, to contaminated livestock trailers. The objective of this study was to test the efficacy of an accelerated hydrogen peroxide (AHP) disinfectant for inactivating PEDV in swine feces on metal surfaces under freezing conditions. One 15.24 X 15.24 X 2.54 cm aluminum coupon, contaminated with swine feces, and randomly matched to one pig was the experimental unit. Eight treatment groups representing two AHP concentrations (1:16 and 1:32) in a 10% propylene glycol solution, two contact times in a -10 °C freezer (40 min and 60 min), and two levels of fecal contamination (5 mL and 10 mL) in addition to negative and positive control groups were evaluated. Forty 3-week-old pigs, intragastrically inoculated with the contents of the coupons after treatment, were used as a bioassay to determine the infectivity of PEDV after treatment. Infectivity was determined by detection of virus with a nucleocapsid (N) gene-based quantitative real-time reverse transcription polymerase chain reaction (RT-qPCR) on rectal swabs collected from the inoculated pigs on days three and seven post-inoculation. RESULTS: All post-treatment swabs from the negative control coupons were negative for PEDV via RT-qPCR. All post-treatment swabs collected from coupons in the AHP disinfectant treatment groups and the positive control group were positive for PEDV via RT-qPCR. For the bioassay, no rectal swabs from pigs in the negative control (0 of 4) or the AHP disinfectant treatment groups (0 of 32) were positive for PEDV. Rectal swabs from all pigs within the positive control group (4 of 4) were positive for PEDV by RT-qPCR. CONCLUSIONS: Under the conditions of this study, 1:16 and 1:32 dilutions of the AHP disinfectant successfully inactivated PEDV in swine feces on metal surfaces when applied at -10 °C with 40 or 60 min of contact time. This study also suggests that a positive RT-qPCR result for PEDV on an environmental sample should be expected when the AHP disinfectant is applied under freezing conditions, but does not necessarily indicate that an infectious dose of PEDV remains after disinfection.

Prevalence and genetic characterization of Toxoplasma gondii in free-range chickens from grocery stores and farms in Maryland, Ohio and Massachusetts, USA.

Chickens are considered important in the epidemiology of Toxoplasma gondii. Chicken hearts (n = 1185) obtained from grocery stores were tested for T. gondii infection. Antibodies to T. gondii were assayed in fluid removed from the heart cavity using the modified agglutination test (MAT) at 1:5, 1:25, and 1:100 dilutions. MAT antibodies were detected in 222 hearts at 1:5 dilution and 8 hearts at 1:25 dilution, but none were positive at 1:100 dilution. Seropositive (n = 230, 19.4%) chicken hearts were bioassayed in mice and seronegative (n = 157) chickens were bioassayed in cats. Viable T. gondii was not isolated from any hearts by bioassays in mice. The 2 cats fed 60 and 97 hearts did not excrete T. gondii oocysts. The results indicate a low prevalence of viable T. gondii in chickens from grocery stores. Molecular typing of 23 archived T. gondii strains isolated from free-range chickens from Ohio and Massachusetts using the 10 PCR-RFLP markers including SAG1, SAG2 (5'-3'SAG2 and altSAG2), SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, and Apico revealed that seven were ToxoDB PCR-RFLP genotype #1, 11 were genotype #2, one was genotype #3, three were genotype #170, and one was mixed genotype. These results indicate that the clonal genotypes #1 (type II), #2 (type III), and #3 (type II variant) are common in free-range chickens.

Validation of the modified agglutination test for the detection of Toxoplasma gondii in free-range chickens by using cat and mouse bioassay.

The modified agglutination test (MAT) is one of the most commonly used tests for the detection of antibodies to Toxoplasma gondii in animal and human sera. The objective of the present study was to evaluate the diagnostic accuracy of the MAT and bioassay in free-range/backyard (FR) chickens (Gallus domesticus). Previously-published T. gondii test results from 2066 chickens from 19 countries were compiled for the present study. The frequency of isolation of T. gondii increased for MAT titres between 1:5 and 1:160, and ranged from 61 to 75% for antibody titres of 1:160, 1:320, and ⩾1:640. Twenty-three cats fed pooled hearts from a total of 802 FR seronegative (MAT, <1:5) chickens from several countries did not excrete oocysts, indicating a high negative predictive value of MAT because FR chickens would have been exposed to many microbes; cats are the most sensitive indicators of T. gondii infection in tissues and can excrete millions of oocysts after ingesting even a few bradyzoites. Of the 29 cats in this study, six cats, fed hearts pooled from 15-122 FR chickens, excreted oocysts; but these identifications were likely related to misidentification or prozone. Results of the present study support the validity of MAT for the detection of T. gondii infection in chickens.