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Worldwide, enterotoxigenic Escherichia coli (ETEC) cause neonatal diarrhea and high mortality rates in newborn calves, leading to great economic losses. In Bavaria, Germany, no recent facts are available regarding the prevalence of virulence factors or antimicrobial resistance of ETEC in calves. Antimicrobial susceptibility of 8713 E. coli isolates obtained from 7358 samples of diseased or deceased diarrheic calves were investigated between 2015 to 2019. Considerably high rates of 84.2% multidrug-resistant and 15.8% extensively drug-resistant isolates were detected. The resistance situation of the first, second and third line antimicrobials for the treatment, here amoxicillin-clavulanate, enrofloxacin and trimethoprim-sulfamethoxazole, is currently acceptable with mean non-susceptibility rates of 28.1%, 37.9% and 50.0% over the investigated 5-year period. Furthermore, the ETEC serotypes O101:K28, O9:K35, O101:K30, O101:K32, O78:K80, O139:K82, O8:K87, O141:K85 and O147:K89, as well as the virulence factors F17, F41, F5, ST-I and stx1 were identified in a subset of samples collected in 2019 and 2020. The substantially high rates of multi- and extensively drug-resistant isolates underline the necessity of continuous monitoring regarding antimicrobial resistance to provide reliable prognoses and adjust recommendations for the treatment of bacterial infections in animals.

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BACKGROUND: Borrelia burgdorferi is a tick-borne spirochete that causes Lyme borreliosis (LB). After an initial tick bite, it spreads from the deposition site in the dermis to distant tissues of the host. It is generally believed that this spirochete disseminates via the hematogenous route. Borrelia persica causes relapsing fever and is able to replicate in the blood stream. Currently the exact dissemination pathway of LB pathogens in the host is not known and controversially discussed. METHODS: In this study, we established a strict intravenous infection murine model using host-adapted spirochetes. Survival capacity and infectivity of host-adapted B. burgdorferi sensu stricto (Bbss) were compared to those of B. persica (Bp) after either intradermal (ID) injection into the dorsal skin of immunocompetent mice or strict intravenous (IV) inoculation via the jugular vein. By in vitro culture and PCR, viable spirochetes and their DNA load in peripheral blood were periodically monitored during a 49/50-day course post-injection, as well as in various tissue samples collected at day 49/50. Specific antibodies in individual plasma/serum samples were detected with serological methods. RESULTS: Regardless of ID or IV injection, DNA of Bp was present in blood samples up to day 24 post-challenge, while no Bbss was detectable in the blood circulation during the complete observation period. In contrast to the brain tropism of Bp, Bbss spirochetes were found in ear, skin, joint, bladder, and heart tissue samples of only ID-inoculated mice. All tested tissues collected from IV-challenged mice were negative for traces of Bbss. ELISA testing of serum samples showed that Bp induced gradually increasing antibody levels after ID or IV inoculation, while Bbss did so only after ID injection but not after IV inoculation. CONCLUSIONS: This study allows us to draw the following conclusions: (i) Bp survives in the blood and disseminates to the host's brain via the hematogenous route; and (ii) Bbss, in contrast, is cleared rapidly from the blood stream and is a tissue-bound spirochete.

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A total of 143 horses were included in a study to test a commercial vaccine against Lyme borreliosis. The vaccine contained three different antigens (outer surface protein A, OspA) to prevent the infection with spirochetes - B.burgdorferi sensu stricto, B. afzelii and B. garinii. Horses in Group A (49 animals) received two vaccinations on days 0 and 14 and a booster on day 365, whereas 50 horses in Group B received an additional booster vaccination on day 180. Group C (44 animals) was not immunized. Total antibody levels and specific OspA antibody responses were assessed quantitatively and qualitatively in two-month intervals over 13-month period. Vaccinees in Groups A and B developed high OspA antibodies levels, whereas horses in Group C did not show specific antibody responses. The additional vaccination applied in Group B enhanced the specific OspA antibody response significantly and prevented its rapid decline.

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Anaplasma phagocytophilum (Ap) is a tick-transmitted obligate intracellular bacterium and the causative agent of the granulocytic anaplasmosis in various species of domestic animals and in humans. During intracellular development Ap transforms from a dense-cored cell form into a reticulate cell form and vice versa. For isolation of intracellular bacteria, a range of different purification methods is used. However, unlike other Gram-negative bacteria Ap is considered to be sensitive to mechanical stress and osmolarity changes. An updated semi-purification method using rock tumbler grit is introduced here to increase the outcome of bacteria and to facilitate the procedure of host cell lysis. The objective of this study was to evaluate the structural integrity and infectivity of Ap after lysis of the host cells using rock tumbler grit and to compare the outcome to that of the frequently used method, syringe lysis. Human promyelocytic leukemia cell lines (HL-60) were infected with Ap and following host cell-free bacteria were assessed by transmission electron microscopy. The outcome of the different purification methods was compared using live/dead-staining based on immunofluorescence to count the number of viable bacteria and real-time PCR to compare the amount of DNA. Subsequently the isolated bacteria were tested to infect naive cell cultures. We observed that both Ap dense-cored cells and reticulate cells are preserved intact after the application of rock tumbler grit. The number of viable, host cell-free bacteria was higher by factor 1.7-2.4 compared to the syringe lysis protocol. Quantitative analysis based on real-time PCR showed an increase of bacterial DNA up to 1.6-2.9 times higher using the rock tumbler grit protocol. Bacteria released from the same number of infected host cells were used for new infections. Flow cytometric analysis of the cell cultures confirmed that the number of Ap organisms recovered by using the rock tumbler grit protocol resulted in higher infection rates than the number of Ap organisms recovered by using syringe lysis protocol. Our observations indicate that the rock tumbler grit protocol can be applied as a safe, robust and convenient method to recover Ap compared to syringe lysis.

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BACKGROUND: Bovine tuberculosis (bTB), which is caused by Mycobacterium bovis and M. caprae, is a notifiable animal disease in Germany. Diagnostic procedure is based on a prescribed protocol that is published in the framework of German bTB legislation. In this protocol small sample volumes are used for DNA extraction followed by real-time PCR analyses. As mycobacteria tend to concentrate in granuloma and the infected tissue in early stages of infection does not necessarily show any visible lesions, it is likely that DNA extraction from only small tissue samples (20-40 mg) of a randomly chosen spot from the organ and following PCR testing may result in false negative results. In this study two DNA extraction methods were developed to process larger sample volumes to increase the detection sensitivity of mycobacterial DNA in animal tissue. The first extraction method is based on magnetic capture, in which specific capture oligonucleotides were utilized. These nucleotides are linked to magnetic particles and capture Mycobacterium-tuberculosis-complex (MTC) DNA released from 10 to 15 g of tissue material. In a second approach remaining sediments from the magnetic capture protocol were further processed with a less complex extraction protocol that can be used in daily routine diagnostics. A total number of 100 tissue samples from 34 cattle (n = 74) and 18 red deer (n = 26) were analyzed with the developed protocols and results were compared to the prescribed protocol. RESULTS: All three extraction methods yield reliable results by the real-time PCR analysis. The use of larger sample volume led to a sensitivity increase of DNA detection which was shown by the decrease of Ct-values. Furthermore five samples which were tested negative or questionable by the official extraction protocol were detected positive by real time PCR when the alternative extraction methods were used. By calculating the kappa index, the three extraction protocols resulted in a moderate (0.52; protocol 1 vs 3) to almost perfect agreement (1.00; red deer sample testing with all protocols). CONCLUSION: Both new methods yielded increased detection rates for MTC DNA detection in large sample volumes and consequently improve the official diagnostic protocol.

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