Natural Co-Exposure to Borrelia burgdorferi s.l. and Anaplasma phagocytophilum: Unraveling the Hematological Profile in Sheep.

The occurrence of co-infected hosts and questing ticks with more than one tick-borne pathogen-as in the case of Anaplasma phagocytophilum and Borrelia burgdorferi sensu lato-is expected in endemic regions. Their synergy-in terms of pathogenesis and disease severity-has been suggested previously in humans. Limited data exist on the clinicopathological alterations in co-infected sheep. In this study, we investigated the impact of A. phagocytophilum and B. burgdorferi s.l. seropositivity, alone and in combination, on the hematological parameters of naturally infected sheep. A complete blood count was performed, and indirect immunofluorescence assays were used to detect IgG antibodies against A. phagocytophilum and IgG and IgM antibodies against B. burgdorferi s.l. Single natural exposure to B. burgdorferi s.l. was characterized by low Packed Cell Volume (PCV) values and platelet (PLT) counts, while single exposure to A. phagocytophilum was characterized by low PCV values, low white blood cell (WBC) counts, and an increased risk for leukopenia and neutropenia. Co-exposure resulted in the most severe blood abnormalities; all the blood parameters decreased, and the sheep presented an increased risk for anemia. Our study showed that natural co-exposure to A. phagocytophilum and B. burgdorferi s.l. in sheep leads to more severe blood abnormalities and enhances the pathogenic processes. More studies are needed to clarify the possible background mechanisms.

Evaluation of Heller's reaction and the urine dipstick method alone or in combination to detect proteinuria in sheep.

BACKGROUND: Urine dipstick and Heller's reaction are easy first-line screening tests for the detection of proteinuria; however, the performance of these methods in alkaline ovine urine is largely unknown. OBJECTIVE: The aim of this study was to evaluate the diagnostic performance of Heller's reaction alone or in combination with dipstick for the detection of proteinuria in sheep, using the urine protein to creatinine ratio (UP/C) with two cut-off values as the reference method. METHODS: Ninety-eight urine samples were collected from sheep using the transient apnea method. Heller's reaction, the dipstick method, and the UP/C ratio were used to assess proteinuria. The results were statistically analyzed twice, based on two different UP/C cut-off values of 0.2 and 0.5. Cohen's kappa value was used to determine the agreement between the UP/C ratios and Heller's reaction, the dipstick method, or the combination of methods. Sensitivity, specificity, and positive and negative likelihood ratios were calculated. ROC curves were also generated, and the areas under the curve (AUC) were evaluated to determine the optimal threshold for the numerical values of the two methods. RESULTS: Heller's reaction is more specific (96.67% and 96.00% when the cut-off value is 0.2 and 0.5, respectively) than the dipstick method, while the dipstick method was more sensitive (91.18% and 91.30%, when the cut-off value was 0.2 and 0.5, respectively) than Heller's reaction for the detection of proteinuria. Both tests were accurate when any grade >0 was considered positive. CONCLUSIONS: Proteinuria can almost be excluded in ovine urine samples with negative Heller's reaction and dipstick test.

Estimation of white blood cell and platelet counts in ovine blood smears, and a comparison with the ADVIA 120 hematology analyzer.

BACKGROUND: Manual evaluation of blood cell counts on stained blood films is a common procedure in resource-limited laboratories of farm animal clinics. Moreover, settings for sheep blood cell counts are not provided on most veterinary hematology analyzers. OBJECTIVES: We aimed to (a) compare the results of white blood cell (WBC) counts evaluated microscopically on ovine blood smears with those obtained by the ADVIA 120 hematology analyzer and validate appropriate correction factors for the manual technique; and (b) assess the two suggested factors to calculate platelet counts on blood smears in sheep. METHODS: The blood samples of 57 sheep were used to generate a regression equation between the average WBC count per field and the WBC count determined using the ADVIA 120 analyzer. Thirty-one new ovine samples were used to assess the agreement between the calculated WBC counts based on a generated equation and those obtained by the analyzer using the Passing-Bablok test and Bland-Altman plots. Similarly, agreements between platelet counts using two different factors for platelet calculation were assessed using the Bland-Altman plot. RESULTS: The average bias of calculated WBC counts was 0.4%, with precision and accuracy being over 95%. Regarding calculated platelet counts, Bland-Altman plots revealed a bias of 26.4% and 1.4% when the average number of platelets per field was multiplied by 15 000 and 20 000, respectively. CONCLUSIONS: Microscopic WBC counting in ovine blood is a reliable alternative to automated analyses using the generated equation. A better agreement between the two methods was observed when a factor of 20 000 was used to calculate platelet counts in ovine blood smears.

Short communication: Bovine mastitis caused by a multidrug-resistant, mcr-1-positive (colistin-resistant), extended-spectrum ß-lactamase-producing Escherichia coli clone on a Greek dairy farm.

We performed a survey aimed at analyzing milk samples collected from cows with mastitis for the presence of extended-spectrum ß-lactamase (ESBL)-producing Escherichia coli. Single-quarter mastitic milk samples obtained from 400 cows in 23 Greek dairy herds with a history of E. coli mastitis were processed for the selective isolation of ESBL-producing E. coli. The antimicrobial susceptibility of the ESBL-producing isolates was analyzed using agar disk diffusion, and minimum inhibitory concentrations of colistin were determined by broth microdilution. We used PCR followed by DNA sequencing to characterize the ß-lactamases and mcr-1 (colistin resistance) genes, and for phylotyping and multilocus sequence typing. We found a total of 89/400 (22.25%) E. coli isolates from 12/23 (52%) farms. Six isolates originating from 6 cows on a single farm were ESBL producers and were resistant to cefquinome, amoxicillin-clavulanic acid, aztreonam, ampicillin, and colistin. Five of these isolates were resistant to trimethoprim-sulfamethoxazole, and 5 to streptomycin. The 6 ESBL producers were mcr-1-positive and carried blaTEM-1 genes; 3 also carried blaCTX-M genes, and 3 carried blaSHV genes. All of the ESBL producers belonged to phylogroup A, multilocus sequence type ST666 (n = 5), or a single locus variant of ST666 (n = 1). To our knowledge, this is the first report of endemic bovine mastitis caused by mcr-1-positive, ESBL-producing E. coli. These results highlight the value of active surveillance of antimicrobial resistance not commonly tested by diagnostic laboratories for the early detection of novel resistant strains.

Ciprofloxacin-resistant Escherichia coli isolated from the intestinal microbiota of goats in Greece in the absence of selective pressure.

The presence of ciprofloxacin resistance commensal Escherichia coli (C-R-Ec) was determined for goats in the absence of selective pressure in Northern and Central Greece. The C-R-Ec was categorized in 3 groups with respect to their phenotypic resistance to other antibiotics as well as the carriage of antibiotic resistance genes. The first group consisted of 7 C-R-Ec that were found also resistant to tetracycline. Among them tet(B) (n = 7), qnr(S) (n = 7), and qnr(B) (n = 3) producers were identified by polymerase chain reaction. The second group consisted of 10 C-R-Ec that were found sensitive to all other antibiotics, and their phenotypic resistance to ciprofloxacin was not attributed to the presence of resistance genes. Finally, the third group consisted of 2 C-R-Ec also resistant to sulfamethoxazole. These strains were not carrying any transferable elements that contribute to resistance either to ciprofloxacin or to sulfamethoxazole. This is the first report of ciprofloxacin-resistant E. coli isolated from goats in Greece.

Leukocyte counts in bronchoalveolar lavage fluid obtained from normal and Maedi-Visna-infected sheep.

BACKGROUND: Bronchoalveolar lavage has proven helpful for the diagnosis of certain ovine diseases of the lungs. There is insufficient data concerning the leukocyte profile of bronchoalveolar lavage fluid (BALF) from MaediVisna infected sheep. OBJECTIVE: The objective of this study was to assess the differential leukocyte profile of BALF associated with Maedi virus infection in sheep and to determine whether cytologic examination of BALF is an effective way to diagnose Maedi or determine the severity of lung lesions. METHODS: BALF and serum samples were analyzed from 400 sheep. Sediment smears of bronchoalveolar lavage were stained with Diff-Quik and examined microscopically to obtain a 200-cell differential cell count. Serum was tested using a commercial kit for Maedi-Visna virus antibodies. Lung samples obtained at the time of slaughter were weighed and examined histologically. RESULTS: Maedi-infected sheep (n=267; seropositive with lung lesions) had a significantly higher percentage of lymphocytes and lower percentage of macrophages in BALF than normal sheep (n=133; seronegative and no lung lesions). These differences were significantly more severe in animals with advanced vs moderate lung lesions. Using classification trees, a cut-off of 13.5% lymphocytes was predictive of Maedi infection and a cut-off of 24.5% lymphocytes was predictive of advanced lung lesions. CONCLUSIONS: Cytologic examination of BALF is useful for the clinical diagnosis of Maedi in sheep and provides important information about the severity of the lung lesions.