Expression Profile of Cytokines and Enzymes mRNA in Blood Leukocytes of Dogs with Leptospirosis and Its Associated Pulmonary Hemorrhage Syndrome.

BACKGROUND: Dogs with leptospirosis show similar organ manifestations and disease course as human patients, including acute kidney injury and pulmonary hemorrhage, making this naturally-occurring infection a good animal model for human leptospirosis. Expression patterns of cytokines and enzymes have been correlated with disease manifestations and clinical outcome in humans and animals. The aim of this study was to describe mRNA expression of pro- and anti-inflammatory mediators in canine leptospirosis and to compare it with other renal diseases to identify patterns characterizing the disease and especially its pulmonary form. METHODOLOGY AND PRINCIPAL FINDINGS: The mRNA abundance of cytokines (IL-1α, IL-1ß, IL-8, IL-10, TNF-α, TGF-ß) and enzymes (5-LO, iNOS) was measured prospectively in blood leukocytes from 34 dogs with severe leptospirosis and acute kidney injury, including 22 dogs with leptospirosis-associated pulmonary hemorrhages. Dogs with leptospirosis were compared to 14 dogs with acute kidney injury of other origin than leptospirosis, 8 dogs with chronic kidney disease, and 10 healthy control dogs. Canine leptospirosis was characterized by high 5-LO and low TNF-α expression compared to other causes of acute kidney injury, although the decreased TNF-α expression was also seen in chronic kidney disease. Leptospirosis-associated pulmonary hemorrhage was not characterized by a specific pattern, with only mild changes noted, including increased IL-10 and decreased 5-LO expression on some days in affected dogs. Fatal outcome from pulmonary hemorrhages was associated with low TNF-α, high IL-1ß, and high iNOS expression, a pattern possibly expressed also in dogs with other forms of acute kidney injury. CONCLUSION: The patterns of cytokine and enzyme expression observed in the present study indicate a complex pro- and anti-inflammatory response to the infection with leptospires. The recognition of these signatures may be of diagnostic and prognostic relevance for affected individuals and they may indicate options for newer therapies targeting the identified pathways.

Postpartum reproductive function: association with energy, metabolic and endocrine status in high yielding dairy cows.

This study evaluated the effect of metabolic, endocrine and energy status on onset of ovarian cycle, days open (DO), and conception at first service in 90 multiparous Holstein cows, housed at a research farm. Dry matter intake, milk yield and body weight were measured daily from Week 2 antepartum (a.p.) to Week 20 postpartum (p.p.). Milk composition was determined four times per week and milk acetone was measured weekly. Blood samples for the determination of glucose, non-esterified fatty acids, cholesterol, creatinine, albumin, urea, beta-hydroxybutyrate, leptin, insulin, insulin-like growth factor-1, growth hormone, 3,5,3'-triiodothyronine (T(3)), and thyroxine (T(4)) were taken 2 weeks a.p., in Weeks 1-16, and 20 p.p. between 7:30 and 9:00 h. The onset of ovarian cycle was specified by weekly gynecological examination and by skim milk progesterone determination by radioimmunoassay (twice per week). Energy balance (EB) traits were calculated and expressed as accumulated negative EB from calving to EB equilibrium, EB nadir (EBN), rate of EB recovery after EBN (EBR), and time from calving to EBN and to EB equilibrium, respectively. The onset of ovarian cycle p.p. was not related to EB. However, a low degree of EBN and a fast EBR were associated with fewer DO, and EB at first service was positively related to conception. High plasma levels of T(3) and T(4) p.p. were associated with an early start of ovarian cycle, and high concentrations of glucose and cholesterol with a short calving to conception interval. Conception at first service was positively related to EB at first service and progesterone concentration 10-13 days after first service. In conclusion, thyroid hormones may play an important role in resumption of ovarian cyclicity p.p., and a good energy status enhances the chance of conception at first service and shortens DO.

Quantitative mRNA analysis of eight bovine 5-HT receptor subtypes in brain, abomasum, and intestine by real-time RT-PCR.

Serotoninergic pathways are involved in economically important bovine gastrointestinal (GI) motility disorders such as displaced abomasum and cecal dilatation/dislocation. The existing research tools to investigate the role of serotoninergic pathways in such disorders in ruminants comprise functional pharmacological methods, e.g., in vitro contractility studies in tissue baths, and electromyographical recordings in vivo. However, no tools for quantification of bovine serotonin receptor [5-hydroxytryptamine receptor (5-HTR)] expression were available so far. This study aimed to develop real-time RT-PCR assays for quantitative mRNA analysis of bovine 5-HTR subtypes. Because the bovine 5-HTR coding sequences (CDSs) were completely unknown, multiple species (human, mouse, and rat) alignment of complete CDS was used for primer design in highly homologous regions. LightCycler real-time RT-PCR assays (partial CDS) for the following bovine 5-HTR subtypes were developed and validated: 5-HTR1A, 5-HTR1B, 5-HTR1D, 5-HTR1F, 5-HTR2A, 5-HTR2B, 5-HTR2C, and 5-HTR4. Intra- and inter-assay coefficients of variation (CV) for the eight established assays were small, ranging from 0.49% to 2.46%. As a first physiological application, 5-HTR mRNA expression levels were measured in brain, abomasum, and intestine of 10 healthy, lactating dairy cows. The 5-HTR expression was quantified by normalization to the housekeeping gene glyceraldehyde-phosphate-dehydrogenase (GAPDH). The 5-HTR subtype expression levels ranged from 0.001% (5-HTR2C in intestine) to 1% 5-HTR/GAPDH (5-HTR1B and 5-HTR4 in intestine). There were high variations of 5-HTR subtype mRNA expression within tissues across receptor subtypes and within receptor subtypes across tissues. In conclusion, accurate real-time RT-PCR assays for quantitative analysis of bovine 5-HTR subtype gene expression were developed and validated.