Validation of an Easy Handling Sample Preparation and Triplex Real Time PCR for Rapid Detection of T. equigenitalis and Other Organisms Associated with Endometritis in Mares.

Isolation and identification of Taylorella equigenitalis, the causative agent of contagious equine metritis, by bacteriology is laborious and does not permit differentiation from the other member of the genus, Taylorella asinigenitalis. Moreover, other organisms such as Klebsiella pneumoniae and Pseudomonas aeruginosa can also cause endometritis in mares and warrant diagnostic detection. Our objectives were to develop a rapid preparation method for field swab samples and to validate this protocol using new multiplex real-time polymerase chain reaction (rtPCR) detection tools for identification of these four pathogens. The complete analytical process from sample preparation to PCR analysis was then evaluated against bacteriology, the World Organisation for Health's (OIE) gold standard method for T. equigenitalis and commonly used for the other three pathogens. The diagnostic sensitivity and specificity of this method, which used direct lysis and a multiplex rtPCR, were 100% and >92%, respectively. This study provided a simple-to-use method for prebreeding screening of mares and stallions.

Influenza A Virus Infection in Cats and Dogs: A Literature Review in the Light of the "One Health" Concept.

Influenza A viruses are amongst the most challenging viruses that threaten both human and animal health. Constantly evolving and crossing species barrier, the emergence of novel zoonotic pathogens is one of the greatest challenges to global health security. During the last decade, considerable attention has been paid to influenza virus infections in dogs, as two canine H3N8 and H3N2 subtypes caused several outbreaks through the United States and Southern Asia, becoming endemic. Cats, even though less documented in the literature, still appear to be susceptible to many avian influenza infections. While influenza epidemics pose a threat to canine and feline health, the risks to humans are largely unknown. Here, we review most recent knowledge of the epidemiology of influenza A viruses in dogs and cats, existing evidences for the abilities of these species to host, sustain intraspecific transmission, and generate novel flu A lineages through genomic reassortment. Such enhanced understanding suggests a need to reinforce surveillance of the role played by companion animals-human interface, in light of the "One Health" concept and the potential emergence of novel zoonotic viruses.

The impact of porcine reproductive and respiratory syndrome virus genetic heterogeneity on molecular assay performances.

The remarkable economic losses due to porcine reproductive and respiratory syndrome (PRRS) have stated the control and eradication of this disease is one of the main issues of swine modern farming. The limited cross-protection of vaccine-induced immunity compelled the adoption of strict biosecurity measures that must be associated with the prompt diagnosis of infection. In our study four RT-PCR methods, a RT-PCR, a SYBR Green I and two hydrolysis probes, were compared to evaluate their respective benefits and disadvantages. One hundred and seventy samples originating from 50 farms located in northern Italy were tested with all assays and performances were evaluated using a Bayesian approach to deal with the absence of a Gold Standard. Sequencing the complete of ORF7, the segment targeted by all methods, allowed a gain of insight into the genetic variability of Italian strains and to investigate the role of mismatches on assay sensitivity. Our study evidenced that methods based only on primers-genome interaction better tolerate PRRSV genetic variability, demonstrating a greater sensitivity (Se): SYBR Green I (Se=98.4%) and RT-PCR (Se=99%) outperform both in-house (Se=71.4%) and commercial (Se=91.7%) probe-based methods. On the other hand, probe-based assays allowed an easier genotyping of PRRSV strains and implementation of the internal control system (IC). Phylogenetic analysis allowed demonstration of a presence of two clades circulating continuously in northern Italy since 1996, when their probable ancestors were collected.

Distribution of Porphyromonas gingivalis fimA genotypes in isolates from subgingival plaque and blood sample during bacteremia

Introduction. Porphyromonas gingivalis is considered as a major etiological agent in the onset and progression of chronic destructive periodontitis. Porphyromonus gingivalis fimA type has been correlated to the virulence potential of the strain; therefore this gene could be involved in the ability of P. gingivalis to reach blood stream. Objective. The classifications of P. gingivalis fimA types will be compared in subgingival plaque and blood samples collected after scaling and root root planing of periodontitis patients. Materials and methods. Fifteen periodontitis patients requiring scaling and root planing were enrolled. P. gingivalis isolates were classed to genotype with fimA type-specific PCR assay. fimA gene was sequenced if the isolate was listed as unclassifiable after PCR technique. Results. Six patients showed positive P. gingivalis bacteremia. The most frequent fimA was fimA type II, followed by Ib, III and IV. In blood strains, type II was followed by IV, Ib and III. Conclusion. Type II was the most frequent genotype in blood samples and in subgingival plaque samples. However, no correlation was found between the frequency of any fimA type with SRP induced bacteremia. P. gingivalis fimA type appears to be conserved within individual patients throughout the times of sample collection. fimA gene sequence results were not in agreement with results of fimA genotyping by PCR.

Introducción. Porphyromonas gingivalis es el principal agente etiológico de la periodontitis. El gen fimA ha sido relacionado con la virulencia del microorganismo, lo cual sugiere la participación de dicho gen en la capacidad del microorganismo para alcanzar el torrente sanguíneo. Objetivo. Estudiar la distribución de los tipos de fimA de P. gingivalis en muestras de placa subgingival y de sangre obtenidas durante bacteriemias después de raspaje y alisado radicular. Materiales y métodos. Se practicó un alisado radicular a 15 pacientes con periodontitis. Se obtuvieron aislamientos clínicos de P. gingivalis de la placa subgingival y durante la bacteriemia inducida por el procedimiento. Para la genotipificación se utilizó la técnica de reacción en cadena de la polimerasa (PCR) específica para fimA. En los aislamientos no clasificables por PCR se realizó secuenciación del gen fimA. Resultados. Seis pacientes fueron positivos para bacteriemia por P. gingivalis. La distribución de fimA evaluada en 30 aislamientos de placa subgingival y de sangre mostró una mayor frecuencia del fimA tipo II de P. gingivalis. En los aislamientos de placa subgingival, la detección de fimA tipo II fue seguida por Ib, III y IV; sin embargo, en los aislamientos de sangre el tipo II fue seguido por los tipos IV, Ib y III. Conclusión. En los aislamientos de sangre y de placa subgingival de pacientes con periodontitis el fimA más frecuente fue el tipo II; no fue posible correlacionar el tipo de fimA con la bacteriemia inducida por el alisado radicular. Los resultados de la secuenciación del gen fimA no concuerdan con los obtenidos por PCR.

Distribution of Porphyromonas gingivalis fimA genotypes in isolates from subgingival plaque and blood sample during bacteremia.

INTRODUCTION: Porphyromonas gingivalis is considered as a major etiological agent in the onset and progression of chronic destructive periodontitis. Porphyromonus gingivalis fimA type has been correlated to the virulence potential of the strain; therefore this gene could be involved in the ability of P. gingivalis to reach blood stream. OBJECTIVE: The classifications of P. gingivalis fimA types will be compared in subgingival plaque and blood samples collected after scaling and root root planing of periodontitis patients. MATERIALS AND METHODS: Fifteen periodontitis patients requiring scaling and root planing were enrolled. P. gingivalis isolates were classed to genotype with fimA type-specific PCR assay. fimA gene was sequenced if the isolate was listed as unclassifiable after PCR technique. RESULTS: Six patients showed positive P. gingivalis bacteremia. The most frequent fimA was fimA type II, followed by Ib, III and IV. In blood strains, type II was followed by IV, Ib and III. CONCLUSION: Type II was the most frequent genotype in blood samples and in subgingival plaque samples. However, no correlation was found between the frequency of any fimA type with SRP induced bacteremia. P. gingivalis fimA type appears to be conserved within individual patients throughout the times of sample collection, fimA gene sequence results were not in agreement with results of fimA genotyping by PCR.

Genotypic characterization of Porphyromonas gingivalis isolated from subgingival plaque and blood sample in positive bacteremia subjects with periodontitis.

AIM: The objective of this study was to investigate clonal relationship among Porphyromonas gingivalis isolated from subgingival plaque and blood samples in positive transient bacteremia subjects with periodontitis. MATERIAL AND METHODS: Unrelated patients with general chronic periodontitis or general aggressive periodontitis requiring scaling and root planing (SRP) were included in the study. Genotyping of each isolate was performed using pulsed field gel electrophoresis technique. Genetic relatedness of strains isolated within an individual or between different patients was determined by dendogram analysis. RESULTS: Following SRP, from 16 patients, seven patients showed positive P. gingivalis bacteremia and nine were negative. Thirty-two strains were isolated from subgingival plaque and blood samples before and during induced transient bacteremia. The majority of the patients harboured one clonal type. Two patients showed different clones in plaque and blood samples suggesting that more than one clone can be found in subgingival plaque. P. gingivalis isolates from periodontitis patients after transient bacteremia following SRP, revealed a high heterogeneity among isolates. CONCLUSION: In 6/16 subjects the same P. gingivalis isolate was found in the blood and in oral cavity. P. gingivalis heterogeneity suggests no association of a unique clonal type with transient bacteremia.

Hypovirulent Listeria monocytogenes strains are less frequently recovered than virulent strains on PALCAM and Rapid' L. mono media.

Several selective media have been developed to detect Listeria monocytogenes contaminated foodstuffs. Polymyxin-acriflavine-LiCl-ceftazidime-aesculin-mannitol (PALCAM) and Oxford media, required for the EN ISO method 11 290-1, are used for the detection of Listeria spp. in 2 days based on the expression of esculinase activity. Selective agar media such as Rapid' L. mono and Agar Listeria according to Ottaviani and Agosti (ALOA), based on the activity of phosphatidylinositol phospholipase C (PI-PLC) that allows the specific detection of L. monocytogenes in 2 days, are also used. However, no medium can assess the level of virulence of L. monocytogenes strains. Using a plaque-forming assay followed by subcutaneous footpad inoculation in mice, 15 virulent, 8 hypovirulent and 17 avirulent strains were discriminated among L. monocytogenes strains mainly originating from food (36/40). Their growth was tested on the four selective media. After 2 days, the number of colony forming units (cfu) of all the virulent strains was significantly superior to the number obtained with avirulent strains on all the four media tested, and superior to the number obtained with hypovirulent strains on PALCAM and Oxford media. These results showed a relationship between the level of virulence of L. monocytogenes strains and their growth on the selective agar media tested. Moreover, 1 out of 8 hypovirulent and 5 out of 17 avirulent strains did not grow on Rapid' L. mono medium, and 1 hypovirulent and 8 avirulent strains grew but did not express PI-PLC activity during the 7 days of incubation. The lack of detection of PI-PLC activity on Rapid' L. mono was not related to a gene mutation since these strains expressed enzymatic activity on ALOA medium, which detected up to 92% of the hypo- and avirulent strains. In contrast, some of these strains without growth or enzymatic activity expression would not be detected with PALCAM and Rapid' L. mono in foodstuffs on the second day.

Reliable ELISAs showing differences between resistant and susceptible lines in hens orally inoculated with Salmonella Enteritidis.

Reliable ELISAs were investigated with the aim to select hen lines resistant to Salmonella Enteritidis and producing high levels of antibodies. In the first experiment, the relation between the humoral response and the bacteriological results was assessed on hens from the Y11 resistant line and the L2 susceptible line, orally inoculated with 10(8) CFU S. Enteritidis per animal. Anti-lipopolysaccharide (LPS) IgG titres were higher but the liver and spleen were less contaminated in hens from the Y11 line than in hens from the L2 line (p = 0.013, 0.031 and 0.026 respectively). In the second experiment, the hens were inoculated orally with 1.7 x 10(8) CFU S. Enteritidis per animal in order to select the ELISA methods showing the more significant differences. ELISAs were based on LPS, flagella, LPS from rough (LPS-R) and smooth strains (LPS-S) and detected IgG and IgM antibodies from sera and yolks. No between-line host response variation was observed in the yolk, with LPS-S and R antigens nor with anti-LPS IgM in the sera. Otherwise, significant differences were encountered between hen lines with the ELISAs performed on the sera detecting anti-LPS IgG, anti-flagella IgG or IgM (p = 0.017, 0.017 and p < 0.001 respectively). When comparing the kinetics of the selected ELISAs, the IgG antibodies against LPS detected between-line variations as early as 1 to 4 weeks pi, whereas with IgG against flagella, the differences were only detected at 1 and 2 weeks pi and with IgM against flagella, the differences were significant at 1, 2, 4 and 8 weeks pi. In conclusion, resistant hen lines producing higher levels of antibodies than the susceptible hen lines may be selected with these ELISAs.