G. Gioia

Highly sensitive multiplex PCR for simultaneous detection and discrimination of Dirofilaria immitis and Dirofilaria repens in canine peripheral blood.

Dirofilaria immitis and Dirofilaria repens are the most common species of filarial nematodes described in the dogs with increasing spread into new geographical areas. The diagnosis of canine dirofilariosis is usually based upon the microscopical detection and identification of circulating microfilariae together with ELISA detection of serum circulating heartworm antigens or antibodies. The identification of the parasite species using the traditional approaches sometimes can be difficult and can lead to misdiagnosis especially on samples from areas where both Dirofilaria are present. In this paper we report a new molecular method based on single-step multiplex PCR to detect and differentiate simultaneously and unequivocally D. immitis and D. repens on DNA extracted from canine peripheral blood. The amplification was performed using a set of primers designed on a portion of the small subunit ribosomal RNA gene of the mitochondrion (12S rDNA). The single-step multiplex PCR here described ensured high (4 mf/ml) sensitivity and specificity with reduced cost and time saving. The multiplex PCR assay represents an additional tool for epidemiological studies and routine disease assessment in areas co-endemic for the two Dirofilaria species.

Identification of suitable endogenous controls and differentially expressed microRNAs in canine fresh-frozen and FFPE lymphoma samples

The elucidation of microRNA (miRNA) expression pattern in canine lymphoma is attractive for veterinary and comparative oncology due to similar genetics, physiology and exposure to environment in dogs and humans. In this work, the expression of a panel of mature miRNAs was quantitated in fresh-frozen and formalin-fixed paraffin-embedded (FFPE) lymph nodes from canine lymphoma. The major findings were: the detection of a panel of miRNAs expressed in canine lymph node; the identification of three suitable endogenous controls (let-7a, miR-16, and miR-26b) by NormFinder and geNorm analysis; the concordance between results obtained from fresh-frozen and FFPE samples; the detection of upregulation of miR-17-5p and miR-181a in B- and T-cell lymphomas respectively. This is the first study aimed to the application of miRNAs analysis in canine lymphoma.

Escherichia coli lipopolysaccharides and Staphylococcus aureus enterotoxin B differentially modulate inflammatory microRNAs in bovine monocytes.

MicroRNAs (miRNAs) are a family of regulatory molecules involved in many physiological processes, including activation of cells of the immune system. This study investigated the effect of Escherichia coli lipopolysaccharide (LPS) and Staphylococcus aureus enterotoxin B (SEB) on the expression of five miRNAs involved in the inflammatory response, including miR-9, miR-125 b, miR-155, miR-146 a and miR-223, in bovine CD14(+) cells (monocytes). Incubation of monocytes with SEB induced down-regulation of miR-155, miR-223 and miR-125 b, but not the anti-inflammatory miRNA miR-146 a. Conversely, incubation with LPS upregulated both miR-155 and miR-146 a. In vitro incubation of isolated CD14(+) bovine monocytes with LPS and SEB elicited different and opposite expression of miRNAs reportedly involved in inflammatory reactions.

Immunophenotype-related microRNA expression in canine chronic lymphocytic leukemia.

MicroRNAs (miRNAs) are posttranscriptional regulatory noncoding RNAs used to profile human hematopoietic tumors. In this study, some mature miRNAs was quantitated in peripheral blood from dogs with chronic lymphocytic leukemia (CLL). Relative expression data were normalised against four endogenous controls (let-7a, miR-17-5p, miR-26b, and miR-223) selected by geNorm analysis. The results revealed distinct miRNA patterns in CLL depending on the immunophenotype. Also in dogs, the different miRNAs expression could reflect developmental lineage and tumor differentiation. The similar genetics, physiology and exposure to environment in dogs and humans make the miRNA expression study in canine CLL attractive for comparative oncology.

Short communication: Genotypic and phenotypic identification of environmental streptococci and association of Lactococcus lactis ssp. lactis with intramammary infections among different dairy farms

Lactococcus species are counted among a large and closely related group of environmental streptococci and streptococci-like bacteria that include bovine mastitis pathogenic Streptococcus, Enterococcus, and Aerococcus species. Phenotypic and biochemical identification methods can be inaccurate and unreliable for species within this group, particularly for Lactococcus spp. As a result, the incidence of Lactococcus spp. on the farm may have been historically underreported and consequently little is known about the clinical importance of this genus as a mastitis pathogen. We used molecular genetic identification methods to accurately differentiate 60 environmental streptococci and streptococci-like bacteria isolated from cows with high somatic cell count and chronic intramammary infection (IMI; >2 somatic cell scores above 4) among 5 geographically distinct farms in New York and Minnesota that exhibited an observed increase in IMI. These isolates were phenotypically identified as Streptococcus uberis and Streptococcus spp. Genetic methods identified 42 isolates (70%) as Lactococcus lactis ssp. lactis, including all 10 isolates originally phenotypically identified as Streptococcus uberis. Antibiotic inhibition testing of all Lc. lactis ssp. lactis showed that 7 isolates were resistant to tetracycline. In the present study, a predominance of Lc. lactis ssp. lactis was identified in association with chronic, clinical bovine IMI among all 5 farms and characterized antimicrobial resistance for treatment therapies. Routine use by mastitis testing labs of molecular identification methods for environmental streptococci and streptococci-like bacteria can further define the role and prevalence of Lc. lactis ssp. lactis in association with bovine IMI and may lead to more targeted therapies.

Rapid differentiation of Dirofilaria immitis and Dirofilaria repens in canine peripheral blood by real-time PCR coupled to high resolution melting analysis.

Dirofilaria immitis and D. repens are the principal causative agents of canine filariosis and, although the number of dogs subjected to specific prevention is increasing, the prevalence of these parasites remains high in many areas of the world. The discrimination between the two Dirofilaria species using the classical diagnostic methods can be difficult and may lead to misdiagnosis especially on samples from areas where both Dirofilaria are present. Over the last years, several molecular methods with higher sensitivity and specificity compared to classical microscopy and ELISA assays were designed. Nevertheless, a need for simple, rapid, and cost-effective molecular protocols to accurately discriminate between D. immitis and D. repens still remains. High resolution melting analysis coupled to real-time PCR (real-time PCR-HRMA) is a widely used technique to target sequence polymorphisms of the same gene in different species without the need to perform DNA sequencing or to use species-specific probes. In this work, a fast and cost-effective real-time PCR-HRMA protocol to detect and differentiate simultaneously and unequivocally D. immitis and D. repens microfilarial DNA extracted from peripheral dog blood samples is described. The present method is simpler to use than most other DNA-based methods and provides comparable discrimination between the two sibling species.

The expression ratio of miR-17-5p and miR-155 correlates with grading in canine splenic lymphoma

In dogs as in humans, microRNAs (miRNAs) play a key role in normal and neoplastic hematopoiesis regulation. The general miRNA expression framework varies among different stages of development and differentiation of tumors, and miRNAs are widely investigated as new molecular tools for cancer diagnosis and classification. Canine lymphomas are currently classified according with the WHO classification, but a comprehensive grading study of clinical samples is still lacking, and molecular tools for quick grading are not yet available. In the present work, a retrospective study of the expression profile of a panel of miRNAs in canine primary splenic lymphomas was performed. The formalin fixed, paraffin embedded (FFPE) lymphoma samples were accurately classified according with the WHO classification, and were analyzed for miRNA expression using stem-loop TaqMan real time RT-PCR. For each miRNA investigated, relative and absolute quantification were performed after selecting the best housekeeping genes using the NormFinder and geNorm algorithms. The results of this study show a diversity in miRNA expression in low (L) grade lymphomas compared to intermediate-high (I-H) grade lymphomas. The molar ratio between miR-17-5p and miR-155 correlated with WHO grading. These results highlight the potential use of miR-17-5p/miR-155 molar ratio as a new molecular tool for grading of canine splenic lymphomas. The data here reported further support the utility of monitoring miRNA expression in canine hematopoietic malignancies diagnosis and prognosis.

Validation of a mycoplasma molecular diagnostic test and distribution of mycoplasma species in bovine milk among New York State dairy farms

Mycoplasma mastitis is a contagious and costly disease of dairy cattle that significantly affects animal health and milk productivity. Mycoplasma bovis is the most prevalent and invasive agent of mycoplasma mastitis in dairy cattle, and early detection is critical. Other mycoplasma have been isolated from milk; however, the role and prevalence of these species as mastitis pathogens are poorly understood. Routine screening of milk for mycoplasma by bacteriological culture is an important component of a farm control strategy to minimize a herd mycoplasma outbreak, but phenotypic methods have limited ability to speciate mycoplasma, affecting how farms and practitioners can understand the role and effect of species other than M. bovis in herd health. Fastidious mycoplasma culture can be lengthy and inconclusive, resulting in delayed or false negative reports. We developed and validated a multitarget PCR assay that can in the same day confirm or reject a presumptive positive mycoplasma culture found upon bacteriological testing of clinical specimens, further discriminate between Acholeplasma and Mycoplasma, and identify M. bovis. Coupled with sequence analysis isolates can be further identified as bovine mycoplasma Mycoplasma arginini, Mycoplasma alkalescens, Mycoplasma canadense, Mycoplasma bovirhinis, Mycoplasma bovigenitalium, Mycoplasma californicum, Acholeplasma laidlawii, and Acholeplasma oculi. Assay validation included analysis of 845 mycoplasma representing these species and 30 additional bacterial species obtained from routine milk submissions to the Quality Milk Production Services from New York State farms and veterinary clinics between January 2012 and December 2015. Among 95 herds, we found 8 different Mycoplasma species and 3 different Acholeplasma species, with an overall prevalence of M. bovirhinis of 1%, A. oculi of 2%, M. arginini of 2%, M. californicum of 3%, M. canadense of 10%, M. bovigenitalium of 10%, A. laidlawii of 11%, M. alkalescens of 17%, and M. bovis of 78%. More than one mycoplasma was found in 14% of the herds tested, and both M. bovis and Acholeplasma were found in 6% of the farms. Incorporation of the validated molecular diagnostic assay into routine bacteriological screening as a supportive confirmation and identification tool will lead to an improved assessment of Mycoplasma and Acholeplasma prevalence data, which will facilitate increased knowledge about the role of these mycoplasma in mastitis.

Antimicrobial susceptibilities and random amplified polymorphic DNA-PCR fingerprint characterization of Lactococcus lactis ssp. lactis and Lactococcus garvieae isolated from bovine intramammary infections

In total, 181 streptococci-like bacteria isolated from intramammary infections (IMI) were submitted by a veterinary clinic to Quality Milk Production Services (QMPS, Cornell University, Ithaca, NY). The isolates were characterized by sequence analysis, and 46 Lactococcus lactis ssp. lactis and 47 Lactococcus garvieae were tested for susceptibility to 17 antibiotics. No resistant strains were found for β-lactam antibiotics widely used in clinical practice (penicillin, ampicillin, and amoxicillin), and all minimum inhibitory concentrations (MIC) were far from the resistance breakpoints. Eight strains had MIC intermediate to cefazolin. The random amplification of polymorphic DNA (RAPD)-PCR fingerprint patterns showed a slightly higher heterogeneity for Lc. lactis ssp. lactis isolates than for Lc. garvieae isolates.

Elaphostrongylus cervi in a population of red deer (Cervus elaphus) and evidence of cerebrospinal nematodiasis in small ruminants in the province of Varese, Italy.

Thirty-one faecal samples were collected from red deer in the northern area of Varese, in the Italian region of Lombardy, between August and October 2008. The animals had either been hunted or accidently killed. Examination for internal parasites showed a prevalence of 45.2% for Elaphostrongylus cervi larvae and species identification was confirmed by polymerase chain reaction (PCR). Ninety-seven faecal samples were also collected from two goat flocks grazing in the same area between December 2007 and May 2008. These showed a prevalence of 74.7% for lungworms. Furthermore, the central nervous systems from five goats and one sheep from this area with a history of neurologically related lameness were examined. Histopathology confirmed E. cervi cerebro-spinal nematodiasis in five cases out of six. This study demonstrates E. cervi transmission from wild to domestic ruminants when the animals graze in the same area, and the possible occurrence of clinical disease in infected goats and sheep associated with high prevalence in deer.