Close up of the allantochorion of an aborted kid due to <i>C. burnetii</i> infection.

<p>The allantochorion of an aborted kid is immunostained to detect the presence of <i>C. burnetii</i> antigen. On the left: Numerous macrophages are present in the necropurulent exudate that covers the allantochorion. Both macrophages (arrow) and sloughed trophoblasts (arrow head) are filled with <i>C. burnetii</i>. On the right: macrophages in the stroma of the allantochorion have phagocytised <i>C. burnetii</i> bacteria (arrow). Bar = 20 µm.</p

Molecular Characterization of <i>Serratia marcescens</i> Strain Isolated from Yellow Mealworms, <i>Tenebrio molitor</i>, in The Netherlands

Insect culture has developed rapidly worldwide; it faces important security and safety control issues, including animal infections and disease development. In the Netherlands, in 2021, a ~30% mortality of mealworms, Tenebrio molitor, occurred at one farm, where over-humid sites in the substrate were observed. Bacterial cultures from both the external and internal partsof fry and larger mealworms were identified by MALDI-TOF to predominantly Serratia marcescens, Staphylococcus xylosus and Staphylococus saprofyticus. Due to the important role of S. marcescens as a potential zoonotic bacterium, we performed a molecular characterization of the isolated strain. Genomic analysis showed a multidrug-resistant S. marcescens isolate carrying a tet (41), aac (6′)-Ic, and blaSST-1 chromosomal class C beta-lactamase-resistantgenes, all located on the chromosome. Additionally, several virulence genes were identified. The phylogenetic tree revealed that the S. marcescens strain from this study was similar to other S. marcescens strains from different ecological niches. Although the entomopathogenic activity was not confirmed, this case demonstrates that T. molitor can act as a reservoir and as an alternative path for exposing clinically important antibiotic-resistant bacteria that can affect animals and humans. It underlines the need to keep management factors optimal, before insects and their products enter the feed and food chain

Comparison of a <i>C. burnetii</i> positive placenta and a <i>C. burnetii</i> negative placenta at parturition.

<p>Top. Formalin-fixed allantochorion of an aborted kid (left) and the allantochorion of a kid from a control goat (right). The intercotyledonary allantochorion of the aborted kid is severely thickened and dull with a yellow-brownish exudate, while the cotyledon (C) shows no obvious macroscopic changes. The allantochorion of the control kid on the right is thin, glistening and transparent. Bottom. Left. Haematoxylin- and eosin-stained section of the intercotyledonary allantochorion of an aborted kid. Notice the severe inflammatory changes in the stroma of the allantochorion. The trophoblast layer is lost with dystrophic calcification of necrotic tissue (arrow) and a purulent exudate (arrowhead). Bar = 200 µm. Right. Histological appearance of a normal allantochorion of a kid from a control goat. The trophoblast layer is intact with a normal appearance of the trophoblast cells (T). Low cellularity in the stroma. E = endothelium of the allantochorion. Bar = 100 µm.</p

Results of <i>C. burnetii</i>-specific PCR using DNA isolated from the indicated tissues taken at necropsy of Coxiella-inoculated goats.

<p>abd. taken: abdominally taken, ext. taken: externally taken, “−”: negative result, “+”: 30<u>≤</u>PCR cycle threshold (Ct)<40, “++”: 20≤Ct<30, “+++”: Ct<20, blank: no sample available.</p><p>Results are measurements at the day of necropsy indicated by days post inoculation and days post-partum. Tissues organised per organ system.supp.</p

Pregnancy outcome and <i>C. burnetii</i>-specific PCR results using DNA isolated from the indicated tissues taken at necropsy of the kids in Experiment II.

<p>nec: necropsy before kidding, abo: abortion, kid: kidding (liveborn kids), “−”: negative result, “+”: 30<u>≤</u>PCR cycle threshold (Ct)<40, “++”: 20≤Ct<30, “+++”: Ct<20, blank: no sample available, * = results of 2 kids shown, third one like other 2.</p><p>Results are measurements at the day of necropsy indicated by days post inoculation and days of gestation. Liveborn kids were euthanised on the day of parturition for necropsy. No significant difference between aborted and liveborn kids was observed.</p

Detection of <i>C. burnetii</i> DNA in vaginal mucus of Coxiella-inoculated goats over time.

<p>Detection of <i>C. burnetii</i> DNA in vaginal mucus of four challenged goats of which a complete sampling sequence was present from inoculation until 119, 126, 140 and 141 days post inoculation (dpi). Goat ID 35: ⧫, 36: ▪, 38: ▴, 40: •. Vaginal mucus samples were taken at the indicated dpi and <i>C. burnetii</i> DNA was measured by PCR. Ct value of 40 is negative, Ct value <40 is positive. Parturition days are indicated as open symbols. Until 42 dpi the four goats were negative. Data indicate that <i>C. burnetii</i> was detected in the vaginal mucus after the first parturition in the group.</p

Results of the comparison of the subcutaneous, oral and nasal infection route in pregnant goats with two doses of <i>Coxiella burnetii</i>.

<p>MID: mouse infective dose; dpi: days post inoculum, “−”: negative result, “+”: 30≤PCR cycle threshold (Ct)<40, “++”: 20≤Ct<30, “+++”: Ct<20.</p

Detection of <i>C. burnetii</i> DNA in the faeces of Coxiella-inoculated goats over time.

<p>Detection of <i>C. burnetii</i> DNA in the faeces of four challenged goats of which a complete sampling sequence was present from inoculation until 119, 126, 140 and 141 days post inoculation (dpi). Goat ID 35: ⧫, 36: ▪, 38: ▴, 40: •. Faecal samples were taken at the indicated dpi and <i>C. burnetii</i> DNA was measured by PCR. Ct value of 40 is negative, Ct value <40 is positive. Parturition days are indicated as open symbols. Up until 42 dpi and on 56 dpi the four goats were negative. At 56 dpi three other goats of the group were positive before their parturition (data not shown). Data indicate that <i>C. burnetii</i> was detected in the faeces after parturition.</p

Overview of the placentome of a Coxiella-inoculated goat.

<p>Top: Scanned haematoxylin and eosin stained section of the placentome of a goat necropsied at 56 dpi (day 131 of pregnancy). No inflammatory reaction in the maternal endometrium (M), the placentome (PL) or the foetal allantochorion (F). MM = myometrium, EG = endometrial glands. Bar = 1 mm. Bottom: Higher magnification of areas A, B and C depicted by the rectangles in the overview. Serial section immunostained for the presence of <i>Coxiella burnetii</i> antigen (brownish colour). A. Foetal allantochorion showing severe swelling of the trophoblast cells caused by the formation of large intracytoplasmic vacuoles. The vacuoles are filled with numerous <i>C. burnetii</i> bacteria. Bar = 200 µm. B. Erythrophagous zone showing similar vacuolation and swelling of the trophoblasts at the base of the foetal villi. Numerous <i>C. burnetii</i> bacteria are seen within the vacuoles of the trophoblasts and in the blood-filled lacuna between the foetal and maternal epithelium (arrow). E = erythrocytes. Bar = 200 µm. C. Placentome. Absence of <i>C. burnetii</i> bacteria in the synepitheliochorial placenta. The maternal epithelium (E) and foetal trophoblasts (T) show no morphological alterations. Bar = 100 µm.</p

Detection of <i>C. burnetii</i> DNA in milk of Coxiella-inoculated goats over time.

<p>Detection of <i>C. burnetii</i> DNA in milk of four challenged goats of which a complete sampling sequence was present from inoculation until 119, 126, 140 and 141 days post inoculation (dpi). Goat ID 35: ⧫, 36: ▪, 38: ▴, 40: •. Milk samples were taken at the indicated days post-partum and <i>C. burnetii</i> DNA was measured by PCR. Ct value of 40 is negative, Ct value <40 is positive. After parturition <i>C. burnetii</i> DNA was detected in the milk until 32 days post-partum.</p