Identification of pathogens in mastitis milk samples with fluorescent in situ hybridization

Traditionally, the bacteriological examination of mastitis milk samples is performed by culture followed by biochemical tests on the cultured bacteria to allow identification of the causative pathogen. Depending on the species involved, this classic identification is time-consuming compared to other techniques such as fluorescent in situ hybridization (FISH), a culture-independent method that utilizes oligonucleotides (labeled with a fluorophore) that are specific to a string of target DNA/RNA. In the current study, the applicability of FISH was evaluated for the detection of mastitis pathogens directly in milk samples. To remove interfering lipids and proteins from mastitis milk samples prior to FISH, a previously published enzymatic treatment with savinase was evaluated. FISH was performed using oligonucleotides specific for Staphylococcus aureus, Streptococcus agalactiae, Streptococcus uberis, Enterococcus faecalis, Enterococcus faecium, Escherichia coli, and Trueperella (Arcanobacterium) pyogenes. The enzymatic pretreatment and the sensitivity of FISH were evaluated using spiked whole milk samples and mastitis milk samples with bacterial loads of less than 10(3) up to 10(8) colony-forming units (CFU)/ml. Bacteria were reliably detected in milk samples with bacterial numbers of 10(6) CFU/ml or higher. However, bacteria present in numbers below 10(6) CFU/ml were not detectable in all cases. The ability of FISH to identify mastitis-causing pathogens directly in milk samples, and therefore earlier than classical culture methods, can supplement the classic diagnostic procedures for mastitis milk samples

Detection of Borrelia burgdorferi sensu-lato-Specific Antibodies in Sera of Canine and Equine Origin—A Comparative Study with Two Line Immunoassays

Lyme borreliosis is a vector-borne disease in humans and animals caused by bacteria from the Borrelia burgdorferi sensu lato complex (Bbsl). The possible transmission of Bbsl from companion animals to humans via ticks makes this disease important in terms of One Health approaches. Thus, early and accurate diagnosis and treatment are of utmost importance. Today’s standard for the detection of specific antibodies against Bbsl is a two-tiered test system based on an ELISA for screening combined with a line immunoassay (LIA) for confirmation. In this study, 200 canine and 200 equine serum samples with known antibody status were tested with two different LIAs (A and B). Results were compared regarding sensitivity, specificity, the diagnostic outcome for dogs and horses, as well as operability of the test. The results for canine serum samples corresponded to 94.0%, making both LIAs a good choice for LB diagnostic in dogs. For equine serum samples, the agreement of both tests was 65.5%, displaying the challenge equine samples still provide in LB diagnostic. Major concerns were the interpretation of the OspA antigen (AG) signal and the use of unspecific (i.e., p100/p83) or too sensitive signals on the LIA. The operability of both LIAs was equally user-friendly. Regarding the tests’ evaluation, the scanning process provided by LIA A was a major advantage considering the comparability of the tests

Morphology of Starch Particles along the Passage through the Gastrointestinal Tract in Laboratory Mice Fed Extruded and Pelleted Diets

Simple Summary Starch is the main carbohydrate source in most lab mouse diets. Its properties are influenced by feed processing. This determines how easily accessible it is to enzymatic digestion in the gastrointestinal tract of animals. In previous studies we have shown that there are differences between pelleted and extruded forms of a maintenance diet fed to mice regarding digestibility and microbiome. To complement these findings, the present study presents a morphological study of the starch particles throughout the passage along the gastrointestinal tract of C57BL/6J mice fed either pellets or extrudate. Samples were stained with Lugol's iodine and examined via stereomicroscope and scanning electron microscope. Starch granules in the pelleted diet are mostly intact and compact, thus autoenzymatic digestion in the small intestine is less efficient than in the more accessible starch granules from the extruded diet. For both diet forms, starch accumulation in the caecum was observed, suggesting selective retention of praecaecally undigested starch for microbial fermentation. These findings allow for unique insights in murine starch digestion that are important to understand the digestive physiology of this species. Diet processing impacts on starch properties, such as the degree of starch gelatinization. This affects digestibility, as shown in laboratory mice fed either a pelleted or an extruded diet. In the present study, the morphology of starch particles throughout the digestive tract of mice was visualized. Thirty-two female C57BL/6J mice were used for a feeding trial. They were fed a commercial maintenance diet for laboratory mice, which was available in pelleted and extruded form, for seven weeks. The mice were sacrificed after the feeding period, and chyme samples were collected from five sites (stomach, anterior and posterior small intestine, caecum, colon). Samples of diets, chyme and faeces were analyzed via stereomicroscopy (stained with Lugol's iodine) and scanning electron microscopy (SEM). The starch granules appeared more compact in the pelleted diet, showing first signs of degradation only in the small intestine. The caecum content of both diets group was intensively stained, particles as well as fluid phase, indicating that it contained mainly starch. The SEM pictures of caecum content showed abundant bacteria near starch particles. This suggests selective retention of prae-caecally undigested starch in the murine caecum, likely the site of microbial fermentation

Management of Candida guilliermondii joint infection in a dog

Background: Candida spp. are dimorphic fungi in the family Cryptococcaceae. Infections with Candida spp. are usually rare conditions in dogs, but immunocompromised patients have a higher risk for developing invasive candidal infections. Case presentation: A 5-year-old male Boxer, positive to Leishmania infantum, was referred to the Veterinary Teaching Hospital of the Department of Veterinary Medicine, University of Perugia, Italy for examination of a non-weight bearing left hind limb lameness of a duration of at least 3 months. During this period, treatment involved systemic anti-inflammatory medications and intra-articular corticosteroid administration. On presentation, clinical examination and radiographic findings were suggestive of cranial cruciate ligament deficiency. To support this diagnosis a stifle arthroscopy was performed: it confirmed a partial rupture of cranial cruciate ligament. Samples culture of synovial fluid and membrane was routinely collected as well, and revealed Candida guilliermondii joint infection. Treatment for the C. guilliermondii joint infection involved systemic anti-fungal therapy, joint lavage and intra-articular administration of antifungal drugs. Lameness improved markedly during this treatment, but lameness did not resolve completely, probably due to cranial cruciate ligament deficiency. Tibial tuberosity advancement (TTA) was chosen in order to treat stifle instability and was performed 4 weeks following cessation of treatment of the C. guilliermondii joint infection. Six month after TTA the dog showed a completely recovery with no lameness. Conclusions: To the authors' knowledge, this is the first case of Candida spp. joint infection reported in dogs. The cause of the progression of the joint C. guilliermondii infection remains unclear but it may be associated with leishmaniasis or intra-articular corticosteroid injections. Treatment with systemic and intra-articular anti-fungal therapies was successful. In the evaluation of hind limb lameness in a chronically immunocompromised dog, it would be advisable to consider also an intra-articular Candida spp. infection

Comparison of Four Commercially Available Point-of-Care Tests to Detect Antibodies against Canine Parvovirus in Dogs

Measuring antibodies to evaluate dogs’ immunity against canine parvovirus (CPV) is useful to avoid unnecessary re-vaccinations. The study aimed to evaluate the quality and practicability of four point-of-care (POC) tests for detection of anti-CPV antibodies. The sera of 198 client-owned and 43 specific pathogen-free (SPF) dogs were included; virus neutralization was the reference method. Specificity, sensitivity, positive and negative predictive value (PPV and NPV), and overall accuracy (OA) were calculated. Specificity was considered to be the most important indicator for POC test performance. Differences between specificity and sensitivity of POC tests in the sera of all dogs were determined by McNemar, agreement by Cohen’s kappa. Prevalence of anti-CPV antibodies in all dogs was 80% (192/241); in the subgroup of client-owned dogs, it was 97% (192/198); and in the subgroup of SPF dogs, it was 0% (0/43). FASTest® and CanTiCheck® were easiest to perform. Specificity was highest in the CanTiCheck® (overall dogs, 98%; client-owned dogs, 83%; SPF dogs, 100%) and the TiterCHEK® (overall dogs, 96%; client-owned dogs, 67%; SPF dogs, 100%); no significant differences in specificity were observed between the ImmunoComb®, the TiterCHEK®, and the CanTiCheck®. Sensitivity was highest in the FASTest® (overall dogs, 95%; client-owned dogs, 95%) and the CanTiCheck® (overall dogs, 80%; client-owned dogs, 80%); sensitivity of the FASTest® was significantly higher compared to the one of the other three tests (McNemars p-value in each comparison: <0.001). CanTiCheck® would be the POC test of choice when considering specificity and practicability. However, differences in the number of false positive results between CanTiCheck®, TiterCHEK®, and ImmunoComb® were minimal

Identification of pathogens in mastitis milk samples with fluorescent in situ hybridization

Traditionally, the bacteriological examination of mastitis milk samples is performed by culture followed by biochemical tests on the cultured bacteria to allow identification of the causative pathogen. Depending on the species involved, this classic identification is time-consuming compared to other techniques such as fluorescent in situ hybridization (FISH), a culture-independent method that utilizes oligonucleotides (labeled with a fluorophore) that are specific to a string of target DNA/RNA. In the current study, the applicability of FISH was evaluated for the detection of mastitis pathogens directly in milk samples. To remove interfering lipids and proteins from mastitis milk samples prior to FISH, a previously published enzymatic treatment with savinase was evaluated. FISH was performed using oligonucleotides specific for Staphylococcus aureus, Streptococcus agalactiae, Streptococcus uberis, Enterococcus faecalis, Enterococcus faecium, Escherichia coli, and Trueperella (Arcanobacterium) pyogenes. The enzymatic pretreatment and the sensitivity of FISH were evaluated using spiked whole milk samples and mastitis milk samples with bacterial loads of less than 10(3) up to 10(8) colony-forming units (CFU)/ml. Bacteria were reliably detected in milk samples with bacterial numbers of 10(6) CFU/ml or higher. However, bacteria present in numbers below 10(6) CFU/ml were not detectable in all cases. The ability of FISH to identify mastitis-causing pathogens directly in milk samples, and therefore earlier than classical culture methods, can supplement the classic diagnostic procedures for mastitis milk samples

Seroprevalence of Borrelia burgdorferi sensu lato and Anaplasma phagocytophilum Infections in German Horses

There are limited data on Lyme borreliosis (LB), a tick-borne disease caused by the Borrelia burgdorferi sensu lato complex, in horses. Seropositivity is not necessarily associated with clinical disease. Data on seropositivity against Borrelia burgdorferi and Anaplasma phagocytophilum in German horses are sparse. Therefore, serum samples from horses (n = 123) suspected of having Lyme borreliosis and clinically healthy horses (n = 113) from the same stables were tested for specific antibodies against Borrelia burgdorferi sensu lato and Anaplasma phagocytophilum. The samples were screened for antibodies against Borrelia burgdorferi (ELISA and an IgG line immunoblot assay). Furthermore, the samples were examined for antibodies against B. burgdorferi and Anaplasma phagocytophilum with a validated rapid in-house test (SNAP® 4Dx Plus® ELISA). The clinical signs of suspect horses included lameness (n = 36), poor performance (n = 19), and apathy (n = 12). Twenty-three percent (n = 26) of suspect horses and 17% (n = 18) of clinically healthy horses were seropositive for having a Borrelia burgdorferi sensu lato infection (p = 0.371), showing that the detection of specific antibodies against B. burgdorferi alone is not sufficient for a diagnosis of equine LB. Anaplasma phagocytophilum seropositivity and seropositivity against both pathogens was 20%/6% in suspect horses and 16%/2% in the clinically healthy population, showing only minor differences (p = 0.108). Unspecific testing for antibodies against B. burgdorferi without clinical suspicion of Lyme borreliosis is not recommended since the clinical relevance of seropositivity against Borrelia burgdorferi sensu lato remains to be elucidated

Detection of Leptospira DNA in urine and presence of specific antibodies in outdoor cats in Germany

Objectives Clinical manifestation of infection with Leptospira species in cats is rare. Nevertheless, cats can develop specific antibodies against the spirochetes after infection. In Canada, Taiwan and the USA it was recently demonstrated that naturally infected cats can also shed DNA from pathogenic Leptospira species in their urine, but the zoonotic potential of infected cats is still unclear. The objective of this study was to demonstrate if outdoor cats in Germany shed DNA from pathogenic Leptospira species in their urine. As a second aim, antibody prevalence was determined. Methods Two hundred and fifteen outdoor cats were prospectively recruited. Urine samples were tested by realtime PCR targeting the lipL32 gene of pathogenic Leptospira species. Antibody titres against eight serovars (Australis, Autumnalis, Bratislava, Canicola, Copenhageni, Grippotyphosa, Pomona, Saxkoebing) belonging to seven serogroups (Australis, Autumnalis, Canicola, Grippotyphosa, Icterohaemorrhagiae, Pomona, Sejroe) were determined by microscopic agglutination test. Results Urine samples from 7/215 cats (3.3%;95% confidence interval [CI] 0.9-5.7) were PCR-positive. Specific antibodies were detected in 35/195 cats (17.9%;95% CI: 12.5-23.3) with titres ranging from 1:100 to 1:6400. Australis, Bratislava and Grippotyphosa were the most common serovars. Conclusions and relevance Outdoor cats in Germany can shed DNA from pathogenic Leptospira species. Therefore, outdoor cats should be considered as a possible source of infection for dogs or humans. Further studies are needed to determine the role of Leptospira species as a cause of disease in cats

A Novel Rapid Sample Preparation Method for MALDI-TOF MS Permits Borrelia burgdorferi Sensu Lato Species and Isolate Differentiation

The genus Borrelia comprises vector-borne bacterial pathogens that can severely affect human and animal health. Members of the Borrelia burgdorferi sensu lato species complex can cause Lyme borreliosis, one of the most common vector-borne diseases in the Northern hemisphere. Besides, members of the relapsing fever group of spirochetes can cause tick-borne relapsing fever in humans and various febrile illnesses in animals in tropical, subtropical and temperate regions. Borrelia spp. organisms are fastidious to cultivate and to maintain in vitro, and therefore, difficult to work with in the laboratory. Currently, borrelia identification is mainly performed using PCR and DNA sequencing methods, which can be complicated/frustrating on complex DNA templates and may still be relatively expensive. Alternative techniques such as matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) are not well established for Borrelia spp., although this technique is currently one of the most used techniques for rapid identification of bacteria in microbiological diagnostic laboratories. This is mainly due to unsatisfactory results obtained by use of simple sample preparation techniques and medium-contamination obscuring the mass spectra. In addition, comprehensive libraries for Borrelia spp. MALDI-TOF MS have yet to be established. In this study, we developed a new filter-based chemical extraction technique that allows measurement of high quality Borrelia spp. spectra from less than 100,000 bacteria per spot in MALDI-TOF MS. We used 49 isolates of 13 different species to produce the largest mass-library for Borrelia spp. so far and to validate the protocol. The library was successfully established and identifies >96% of used isolates correctly to species level. Cluster analysis on the sum spectra was applied to all the different isolates, which resulted in tight cluster generation for most species. Comparative analysis of the generated cluster to a phylogeny based on concatenated multi-locus sequence typing genes provided a surprising homology. Our data demonstrate that the technique described here can be used for fast and reliable species and strain typing within the borrelia complex

The Evolution of the Satratoxin and Atranone Gene Clusters of Stachybotrys chartarum

Stachybotrys chartarum is frequently isolated from damp building materials or improperly stored animal forage. Human and animal exposure to the secondary metabolites of this mold is linked to severe health effects. The mutually exclusive production of either satratoxins or atranones defines the chemotypes A and S. Based upon the genes (satratoxin cluster, SC1-3, sat or atranone cluster, AC1, atr) that are supposed to be essential for satratoxin and atranone production, S. chartarum can furthermore be divided into three genotypes: the S-type possessing all sat- but no atr-genes, the A-type lacking the sat- but harboring all atr-genes, and the H-type having only certain sat- and all atr-genes. We analyzed the above-mentioned gene clusters and their flanking regions to shed light on the evolutionary relationship. Furthermore, we performed a deep re-sequencing and LC-MS/MS (Liquid chromatography–mass spectrometry) analysis. We propose a first model for the evolution of the S. chartarum genotypes. We assume that genotype H represents the most ancient form. A loss of the AC1 and the concomitant acquisition of the SC2 led to the emergence of the genotype S. According to our model, the genotype H also developed towards genotype A, a process that was accompanied by a loss of SC1 and SC3