Tick-borne diseases in Europe: Current prevention, control tools and the promise of aptamers.

In Europe, tick-borne diseases (TBDs) cause significant morbidity and mortality, affecting both human and animal health. Ticks can transmit a wide variety of pathogens (bacteria, viruses, and parasites) and feed on many vertebrate hosts. The incidence and public health burden of TBDs are tending to intensify in Europe due to various factors, mainly anthropogenic and often combined. Early detection of tick-borne pathogens (TBPs), preventive measures and treatment are of great importance to control TBDs and their expansion. However, there are various limitations in terms of the sensitivity and/or specificity of detection and prevention methods, and even in terms of feasibility. Aptamers are single-stranded DNA or RNA that could address these issues as they are able to bind with high affinity and specificity to a wide range of targets (e.g., proteins, small compounds, and cells) due to their unique three-dimensional structure. To date, aptamers have been selected against TBPs such as tick-borne encephalitis virus, Francisella tularensis, and Rickettsia typhi. These studies have demonstrated the benefits of aptamer-based assays for pathogen detection and medical diagnosis. In this review, we address the applications of aptamers to TBDs and discuss their potential for improving prevention measures (use of chemical acaricides, vaccination), diagnosis and therapeutic strategies to control TBDs.

Anaplasma phagocytophilum in urban and peri-urban passerine birds in Ile-de-France.

Wild animals in general, birds in particular, play a key role in transporting ticks and propagating tick-borne pathogens. Several studies have confirmed the infection of birds with Anaplasma phagocytophilum, with overall prevalence varying widely from country to country and/or study to study. This zoonotic bacterium, transmitted mainly by ticks of the genus Ixodes, is responsible for granulocytic anaplasmosis in humans (HGA) and domestic animals (cats, dogs, horses). The disease is also called tick-borne fever (TBF) in ruminants. Extremely rare in the USA, TBF is very common in Europe, where it causes economic losses in livestock. Conversely, HGA is well established in the USA whereas only a few less severe cases have been observed in Europe. Current typing techniques support the existence of multiple variants with differences in virulence/pathogenicity and tropism for certain tick and host species. However, epidemiological cycles remain difficult to characterize in Europe. Several studies describe a cycle apparently involving only birds in Europe, but no such study has been conducted in mainland France. Our objectives were to search for A. phagocytophilum in passerine birds in the Ile-de-France region and to explore their diversity using groEL and ankA gene typing and multilocus sequence typing (MLST). Various tissues (spleen, liver, and skin) were collected from cadavers of 680 passerines between March and December 2021. The presence of A. phagocytophilum was detected by qPCR Taqman targeting the msp2 gene. Three blackbirds (Turdus merula) were found positive, representing detection rates of 0.4 % in all birds tested and 3.3 % in blackbirds. The higher frequency of detection in blackbirds could be at least partially explained by their lifestyle, as they feed on the ground. Analysis of the results of groEL and ankA typing and MLST from positive blackbirds support the hypothesis that the avian A. phagocytophilum strains in Ile-de-France are distinct from those found in mammals, and that they form their own cluster in Europe.

Aptamer selection against cell extracts containing the zoonotic obligate intracellular bacterium, Anaplasma phagocytophilum.

A. phagocytophilum is a zoonotic and tick-borne bacterium, threatening human and animal health. Many questions persist concerning the variability of strains and the mechanisms governing the interactions with its different hosts. These gaps can be explained by the difficulty to cultivate and study A. phagocytophilum because of its strict intracellular location and the lack of specific tools, in particular monoclonal antibodies, currently unavailable. The objective of our study was to develop DNA aptamers against A. phagocytophilum, or molecules expressed during the infection, as new study and/or capture tools. Selecting aptamers was a major challenge due to the strict intracellular location of the bacterium. To meet this challenge, we set up a customized selection protocol against an enriched suspension of A. phagocytophilum NY18 strain, cultivated in HL-60 cells. The implementation of SELEX allowed the selection of three aptamers, characterized by a high affinity for HL-60 cells infected with A. phagocytophilum NY18 strain. Interestingly, the targets of these three aptamers are most likely proteins expressed at different times of infection. The selected aptamers could contribute to increase our understanding of the interactions between A. phagocytophilum and its hosts, as well as permit the development of new diagnostic, therapeutic or drug delivery appliances.

Optimized Lambda Exonuclease Digestion or Purification Using Streptavidin-Coated Beads: Which One Is Best for Successful DNA Aptamer Selection?

The high failure rate of the in vitro aptamer selection process by SELEX (Systematic Evolution of Ligands by EXponential enrichment) limits the production of these innovative oligonucleotides and, consequently, limits their potential applications. The generation of single-stranded DNA (ssDNA) is a critical step of SELEX, directly affecting the enrichment and the selection of potential binding sequences. The main goal of this study was to confirm the best method for generating ssDNA by comparing the purification of ssDNA, using streptavidin-coated beads, and lambda exonuclease digestion, and by improving ssDNA recovery through protocol improvements. In addition, three techniques for quantifying the ssDNA generated (Qubit vs. NanodropTM vs. gel quantification) were compared, and these demonstrated the accuracy of the gel-based quantification method. Lambda exonuclease digestion was found to be more efficient for ssDNA recovery than purification using streptavidin-coated beads, both quantitatively and qualitatively. In conclusion, this work provides a detailed and rigorous protocol for generating ssDNA, improving the chances of a successful aptamer selection process.

Novel Chronic Anaplasmosis in Splenectomized Patient, Amazon Rainforest.

We report a case of unusual human anaplasmosis in the Amazon rainforest of French Guiana. Molecular typing demonstrated that the pathogen is a novel Anaplasma species, distinct to all known species, and more genetically related to recently described Anaplasma spp. causing infections in rainforest wild fauna of Brazil.

Red blood cell abnormalities occur in dogs with congenital ventricular outflow tract obstruction.

OBJECTIVE: To document RBC abnormalities in dogs with congenital ventricular outflow tract obstruction. ANIMALS: 62 dogs with pulmonic stenosis (PS) or aortic stenosis (AS) and 20 control dogs were recruited. PROCEDURES: The proportions of RBCs that were schistocytes, acanthocytes, and keratocytes were assessed. Complete blood cell counts were performed. Tested variables included hemoglobin concentration, hematocrit, and erythrocyte count. RESULTS: Median (interquartile range [IQR]) peak systolic Doppler-derived trans-stenotic pressure gradient (∆P) values were 161 mm Hg (108 to 215 mm Hg) and 134 mm Hg (125 to 165 mm Hg) for dogs with PS and AS, respectively. Hematologic abnormalities were detected in most dogs with AS or PS (54/62 [87%]) versus 8/20 [40%] in control dogs, with schistocytes found in 40 of 62 (65%; median, 0.1% RBCs; IQR, 0% to 0.3%), acanthocytes in 29 of 62 (47%; median, 0.3% RBCs; IQR, 0% to 0.9%), keratocytes in 39 of 62 (63%; median, 0% RBCs; IQR, 0% to 0.2%), and hemolytic anemia in 4 dogs with PS. No significant association was identified between these abnormalities and ∆P. However, 3 of 4 dogs with anemia had a ∆P > 200 mm Hg (range, 242 to 340 mm Hg). The dog with the highest ∆P value also had the most severe anemia and schistocytosis, and both resolved after balloon valvuloplasty. CLINICAL RELEVANCE: Poikilocytosis is common in dogs with congenital ventricular outflow tract obstruction, with anemia only observed in few dogs with high ∆P values.

Assessment of urinary 15-F2 -isoprostanes in dogs with urothelial carcinoma of the urinary bladder and other lower urinary tract diseases.

BACKGROUND: The 15-F2 -isoprostanes are by-products of oxidative stress and are increased in the urine of people with lower urinary tract diseases (LUTD), especially urinary neoplasia. Urothelial carcinoma (UC) is the most common urinary neoplasm in dogs. Earlier detection of UC by noninvasive means could lead to improved outcomes. Urinary 15-F2 -isoprostanes potentially could provide this means, but have not been evaluated in dogs with UC. OBJECTIVE: The objective of this study was to measure urinary 15-F2 -isoprostanes in dogs with UC and dogs with other LUTD. ANIMALS: One hundred seventeen dogs: 46 dogs with UC, 30 dogs with LUTD, and 25 control dogs. METHODS: Any dog that was presented with dysuria was eligible for inclusion. Diagnosis of UC was confirmed histologically. Urinalysis was performed in each case, and 15-F2 -isoprostanes quantified by gas chromatography-negative ion chemical ionization-mass spectrometry (GC-NICI-MS) and normalized to urinary creatinine concentration. RESULTS: Dogs with urinary diseases (UC + LUTD) had higher median urinary 15-F2 -isoprostanes when compared to control dogs (5.92 ng/mg [range, 0.46-31.03] vs 3.73 [range, 1.8-7.98]; P = .02). Urinary 15-F2 -isoprostanes were similar in dogs with UC (5.33 ng/mg [range, 0.46-31.03]) compared to dogs with LUTD (6.29 ng/mg [range, 0.54-18.93]; P = .47) and control dogs (P = .06). Dogs with UC had higher qualitative measures of proteinuria (P = .004), hematuria (P = .01), and epithelial cells on urinalysis (P = .002) compared to the other groups. CONCLUSIONS AND CLINICAL IMPORTANCE: Urinary F2 -isoprostanes are not useful for the detection of UC in dogs. Future research could evaluate urinary 15-F2 -isoprostanes as a marker of inflammation in disease progression and prognosis.

Feline urinary F2-isoprostanes measured by enzyme-linked immunoassay and gas chromatography-mass spectroscopy are poorly correlated.

15-F2T-isoprostanes are byproducts of lipid peroxidation and were determined to be the best marker of oxidative injury in a rodent model of oxidative stress. A previous study compared methods for measurement of urinary F2-isoprostanes (gas chromatography and negative ion chemical ionization-mass spectrometry, GC-NICI-MS; and ELISA) and found poor agreement in dogs, horses, and cows. Surprisingly, fair agreement between these methods was identified in a small population of cats. We evaluated the agreement between GC-NICI-MS and ELISA of urinary F2-isoprostanes in the urine of 50 mature cats ranging from healthy to systemically ill. All urine samples had detectable levels of F2-isoprostanes by both methods. Significant proportional bias and poor agreement were identified between the 2 methods (ρ = 0.364, p = 0.009) for all cats, and in subgroup analysis based on health status. The concentration of urinary F2-isoprostanes was significantly lower in systemically ill cats compared to healthy cats when measured by ELISA (p = 0.002) but not by GC-NICI-MS (p = 0.068). Our results indicate that GC-NICI-MS and ELISA have poor agreement when measuring urinary F2-isoprostanes in cats.

Phase I/II clinical trial of the targeted chemotherapeutic drug, folate-tubulysin, in dogs with naturally-occurring invasive urothelial carcinoma.

PURPOSE: The purpose was to determine the safety and antitumor activity of a folate-tubulysin conjugate (EC0531) in a relevant preclinical animal model, dogs with naturally-occurring invasive urothelial carcinoma (iUC). Canine iUC is an aggressive cancer with high folate receptor (FR) expression similar to that in certain forms of human cancer. EXPERIMENTAL DESIGN: A 3+3 dose escalation study of EC0531 (starting dose 0.2 mg/kg given intravenously at two-week intervals) was performed in dogs with iUC expressing high levels of FRs (>50% positive tumor cells). Pharmacokinetic (PK) analysis was performed, and the maximum tolerated dose (MTD) was determined. The dose cohort at the MTD was expanded to determine antitumor activity. RESULTS: The MTD of EC0531 was 0.26 mg/kg every two weeks, with grade 3-4 neutropenia and gastrointestinal toxicity observed at higher doses. Treatment at the MTD was well tolerated. Clinical benefit was found in 20 of 28 dogs (71%), including three dogs with partial remission and 17 dogs with stable disease. Plasma EC0531 concentrations in the dogs far exceeded those required to inhibit proliferation of FR-expressing cell in vitro. Unlike human neutrophils, canine neutrophils were found to express FRs, which contributes to the neutropenia at higher doses of EC0531 in dogs. CONCLUSION: EC0531 was well tolerated and had good antitumor activity in dogs with iUC. It is likely that humans will tolerate higher, potentially more effective doses of folate-tubulysin without myelotoxicity because of the absence of FRs on human neutrophils. The results clearly justify the evaluation of folate-tubulysin in human clinical trials.

Pelger-Huët anomaly in a cat.

A 14-year-old, spayed female Domestic Shorthair cat was referred to the Purdue University Veterinary Teaching Hospital (PUVTH) for iodine 131 treatment of hyperthyroidism. Upon arrival, a biochemistry profile and a CBC were performed. Approximately 50% of the neutrophils and all the eosinophils observed were hyposegmented with a mature, condensed chromatin pattern. Nuclei had a band to "dumbbell" shape, and rarely a round shape, suggesting a Pelger-Huët anomaly or a pseudo Pelger-Huët. Based on both a negative FeLV and FIV tests, the absence of any clinical signs to support an inflammatory process, and the persistence of this granulocytic morphology 6 months after its previous admission to the PUVTH, a diagnosis of Pelger-Huët anomaly was established in this cat.

Changes in hematology measurements in healthy and diseased dog blood stored at room temperature for 24 and 48 hours using the XT-2000iV analyzer.

BACKGROUND: Changes in canine hematology measurements may occur when analyses are delayed due to shipment of specimens to a laboratory. OBJECTIVE: The purpose of this study was to report changes in hematologic variables in healthy and diseased canine blood measured with a Sysmex XT-2000iV during storage at room temperature for 24 and 48 hours. METHODS: EDTA-K3 blood samples from 42 healthy and diseased dogs were measured on a Sysmex XT-2000iV analyzer within one hour of sampling, and after storage for 24 and 48 hours at room temperature in the dark. RESULTS: Storage caused little or no change in RBC count, HGB concentration and MCH, while there was a moderate increase in HCT, MCV and reticulocytes count, and a moderate decrease in MCHC. Decreased platelet counts by impedance (PLT-I) and optical (PLT-O) measurements were associated with increased mean platelet volume (MPV), platelet-large cell ratio (P-LCR) and platelet distribution width (PDW), including a right shift in the platelet histogram and a dispersion of the platelet dot plot on the scattergram. The total and differential WBC count remained stable except for decreased monocyte counts. In the scatterplots, monocytes shifted into the lymphocyte population after 24 hours, and neutrophil population shifted to the right appearing in the eosinophil gate at 48 hours of storage. The disease status had only a small effect on storage-induced changes, and observed changes had no consequences for clinical decisions. CONCLUSIONS: Blood storage at room temperature was accompanied by moderate variations in some hematologic variables, awareness of which helps in avoiding misinterpretations.

Complete Genome Sequence of Mycoplasma ovis Strain Michigan, a Hemoplasma of Sheep with Two Distinct 16S rRNA Genes.

We report the complete genome sequence of Mycoplasma ovis strain Michigan. Its single circular chromosome has 702,511 bp and contains 2 copies of the 16S rRNA gene, one corresponding to M. ovis and the other to "Candidatus Mycoplasma haemovis." All housekeeping genes and the 5S-23S rRNA genes are present in single copies.