Erysipelas, a disease caused by Erysipelothrix rhusiopathiae (ER), is an increasing problem in laying hens housed in cage-free systems. This study aimed to monitor immune responses during ER infection of naïve chickens and chickens vaccinated intra muscularly with a commercial inactivated ER vaccine. Chickens were infected intra muscularly with ER at 30 days of age and blood leukocyte counts, serum levels of mannose binding lectin (MBL) and ER-specific IgY were monitored until the experiment was terminated at day 15 after infection. ER was detected in blood from more chickens and at higher bacterial counts in the naïve group (day 1: 1 of 7 chickens; day 3: 6 of 6 chickens) than in the vaccinated group (day 1: 0 of 7 chickens; day 3: 1 of 6 chickens). During the acute phase of infection transient increases in circulating heterophil numbers and serum MBL levels were detected in all ER infected chickens but these responses were prolonged in chickens from the naïve group compared to vaccinated chickens. Before infection IgY titers to ER in vaccinated chickens did not differ significantly from those of naïve chickens but vaccinated chickens showed significantly increased IgY titers to ER earlier after infection compared to chickens in the naïve group. In conclusion, the ER infection elicited prompt acute innate responses in all chickens. Vaccinated chickens did not have high IgY titers to ER prior to infection but did however show lower levels of bacteraemia and their acute immune responses were of shorter duration.

BackgroundBrucella abortus is a highly pathogenic zoonotic agent, tempting for the development of a rapid diagnostic method to enable adequate treatment and prevent further spread. Enrichment of the bacteria is often used as a first step in diagnostics to increase the bacterial number above the detection limit of the real-time PCR. The enrichment of Brucella spp. takes at least 3 days, which might be avoidable if sensitive PCR methods can be used. Since many matrices contain PCR inhibitors, the limit of detection (LOD) must be determined for each separate matrix.Another aim of this study was the determination of survival of Brucella abortus in the analyzed matrices.MethodsThe LOD for the detection of B. abortus in 14 matrices, relevant for human medicine, veterinary medicine and food and feed safety, was determined to evaluate the need of a pre-enrichment step prior to real-time PCR.The survival of B. abortus in the spiked matrices was tested by plate count in a 7-day interval for 132 days.ResultsThe limit of detection for B. abortus in most matrices was in the range of 103–104 CFU/g for cultivation and 104–105 CFU/g for direct real-time PCR.The survival time of B. abortus was less than 21 days in apple purée and stomach content and 28 days in water while B. abortus remained viable at day 132 in milk, blood, spinach and minced meat.ConclusionsA direct PCR analysis without enrichment of bacteria saves at least 3 days. However, the limit of detection between direct PCR and plate count differs in a 10 fold range. We conclude that this lower sensitivity is acceptable in most cases especially if quick analysis are required.

Livestock manure is a valuable source of nutrients for crop production, but can also pose a public health hazard and have negative environmental impacts. This study investigated manure management practices among urban and peri-urban livestock keepers in Cambodia, to identify risk behaviours and socio-economic aspects associated with the handling of manure. A survey including 204 households was conducted, using a structured questionnaire with questions on demographics, socio-economic characteristics and household practices related to manure management. Faecal samples were obtained from pig pens and pig manure storage units for analysis of the potential zoonotic pathogens Salmonella enterica (Polymerase Chain Reaction (PCR)), Ascaris suum and Trichuris suis (McMaster flotation technique). The survey revealed a difference in management between cattle and pig manure. Cattle manure was most commonly used as fertiliser for crop production (66%) (p<0.001), whereas pig manure was most commonly dumped in the environment (46%) (p<0.001). Logistic regression models showed that households with a lower socio-economic position were more likely to dump pig manure (p<0.001), with scarcity of agricultural land (p<0.001) and lack of carts for transportation of manure (p<0.01) being identified as contributing factors. Salmonella enterica was detected in 9.7% of manure samples, while Ascaris suum and Trichuris suis were detected in 1.6% and 2.4% of the samples, respectively. The results presented in this study indicate that manure management by urban and peri-urban households may pose a public health threat and an environmental hazard. There is evidently a need for further knowledge support to the livestock keepers to promote good management practices.

Purpose The present study aimed to establish pretreatment protocols as well as real-time and droplet digital polymerase chain reaction (PCR) methodologies to detect and quantify Erysipelothrix rhusiopathiae (ER) DNA in blood samples from infected chickens, as tools for routine diagnostics and monitoring of experimental infections. Chicken blood is a problematic matrix for PCR analysis because nucleated erythrocytes contribute large amounts of host DNA that inhibit amplification.Methodology Using artificially spiked samples of fresh chicken blood, as well as blood samples from three experimental infection studies, the performance of pretreatment protocols, including choice of blood stabilization agent, centrifugation speeds and Ficoll gradient separation, was evaluated. The results were compared with those from traditional culture-based protocols combined with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS).Results/Key findings Simple preparations producing cell-free samples performed well on artificial spike-in samples, providing high sensitivity. However, performance was poor in clinical samples or artificial samples where the bacteria were incubated for 4 h or more in fresh blood prior to DNA extraction. In these samples, a Ficoll separation protocol that creates samples rich in lymphocytes, monocytes and thrombocytes prior to DNA extraction was far more effective.Conclusions Our results indicate that ER bacteria undergo rapid phagocytosis in chicken blood and that analysis of a blood fraction enriched for phagocytic cells is necessary for reliable detection and quantification. The presented results explain the poor performance of PCR detection reported in previously published experimental ER infection studies, and the proposed solutions are likely to have broader implications for PCR-based veterinary diagnostics in non-mammalian host species such as poultry and fish.

Infectious keratoconjunctivitis (IKC), the most common ocular disease in ruminants worldwide, has affected semi-domesticated Eurasian reindeer (Rangifer tarandus tarandus) for over 100 years, both as individual cases and in outbreaks affecting tens to hundreds of animals. Recurrent IKC outbreaks have been affecting a semi-domesticated reindeer herd in Östra Kikkejaure (Norrbotten county, Sweden) from 2014. The latest episode of these recurrent outbreaks, in winter 2016/2017, was investigated in this study. Clinical findings were in line with previous reports of IKC in semi-domesticated reindeer and the clinical signs displayed by the affected animals (n = 30) included increased lacrimation, follicular conjunctivitis, purulent secretions around the affected eyes and corneal edema. Laboratory analyses of the samples revealed the presence of Chlamydiaceae in most samples obtained from the clinically affected animals (98.3%, n = 60), but also a high seroprevalence of cervid herpesvirus 2 (CvHV2) antibodies (56.6%, n = 53). Moraxella bovoculi was isolated from nine IKC-affected animals during the outbreak (45.0%, n = 20). All affected animals were treated with long-acting antibiotics and recovered from the disease, testing negative for the presence of Chlamydiaceae DNA by PCR 16 days and 3 months after the initial treatment. For the first time, Chlamydia pecorum was identified in semi-domesticated reindeer, and the involvement of Chlamydiaceae in a clinical outbreak of IKC is reported. The CvHV2 seroprevalence (56.6%) and the data obtained from a previous outbreak in 2014 also suggest the involvement of the reindeer alphaherpesvirus in the recurrent outbreaks.