Effect of repeated intravenous lipopolysaccharide infusions on systemic inflammatory response and endometrium gene expression in Holstein heifers.

This study aimed to evaluate the effect of repeated intravenous lipopolysaccharide (LPS) infusions in nonlactating heifers on (1) the systemic proinflammatory state as measured by biomarkers in blood and plasma, and (2) endometrial gene expression of candidate transcripts on d 15 of gestation. Our hypothesis was that target transcripts related to a major functional group would be negatively modified in the preimplantation endometrium by the LPS treatments. In the first experiment (n = 13), a systemic proinflammatory state [defined as increased plasma concentrations of tumor necrosis factor (TNF)-α and haptoglobin for 2 wk] was established using 2 different sequential LPS infusion protocols. In the second experiment, heifers (n = 22; 11 mo of age) had their time of ovulation synchronized by a modified Ovsynch protocol and were enrolled in 1 of 2 treatments: control (CON; n = 11), which received sterile saline solution i.v., and LPS treatment (LPS; n = 11), submitted to repeated i.v. LPS injections (0.10, 0.25, 0.50, 0.75, 1.00, and 1.25 µg/kg) starting 2 d after artificial insemination (AI; d 0) and then every other day until d 15 after AI. At each LPS injection, rectal temperatures were measured hourly for 6 h. Blood samples were collected from d -1 to d 13 for analyses of progesterone, TNF-α, and haptoglobin in plasma, along with white blood cell (WBC) count and differential analysis. On d 15, endometrium tissue biopsies were taken and kept at -80°C until quantitative real-time PCR analysis of 30 target transcripts related to the immune system, adhesion molecules, and endometrium receptivity. Data were checked for normality and analyzed by repeated-measures ANOVA using PROC UNIVARIATE and PROC MIXED of SAS (SAS Institute Inc., Cary, NC). After each LPS injection, temperature was greater in the first 4 h in the LPS group compared with CON. Both TNF-α and haptoglobin increased in the LPS treatment with a significant treatment by day interaction. Total leukocyte count did not differ between treatments, but the differential count increased for neutrophils, band cells, and monocytes, and decreased for lymphocytes and eosinophils in LPS compared with CON. Progesterone concentrations in plasma did not differ between treatments during the experimental period. Out of 30 target genes analyzed, 3 transcripts were differentially expressed: indoleamine 2,3-dioxygenase (IDO; fold-change = 0.48) and pentraxin-3 (PTX3; fold-change = 0.38) were downregulated, whereas myxovirus-resistance protein (MX1; fold-change = 2.85) was upregulated in the LPS group. Sequential LPS injections were able to induce a prolonged systemic proinflammatory state, but effects on gene expression were limited to transcripts associated with the immune system. These results suggest that a mechanism for subfertility is linked to a proinflammatory state in dairy heifers.

Intramammary infusion of lipopolysaccharide promotes inflammation and alters endometrial gene expression in lactating Holstein cows.

The objective of the current study was to evaluate the effects of 2 intramammary infusions of lipopolysaccharide (LPS) on inflammatory and reproductive parameters and endometrial gene expression of lactating Holstein cows. At 35 ± 7 d in milk, 20 cows were submitted to a Double Ovsynch program and randomly assigned to control (n = 11) and LPS (n = 9) treatments. Cows from the LPS treatment received 2 intramammary infusions of 25 µg of LPS after morning milking on d 5 and 10 post-AI, whereas control cows were infused with only saline. Blood samples were taken and ultrasound scanning of the ovaries was performed during the entire study before and after AI to determine haptoglobin, tumor necrosis factor-α, and progesterone concentrations as well as response to the hormonal protocol and corpus luteum diameter. Milk yield was evaluated and samples were taken for somatic cell count at 0, 10, 24, 34, and 96 h relative to each infusion. Rumen-reticular temperature was recorded using a rumen-reticular bolus logger and summarized hourly. On d 15 post-AI, uterine flushing for conceptus recovery and endometrial biopsies were performed. Samples of endometrium from cows with positive embryo recovery (control = 5; LPS = 6) were submitted to mRNA extraction and quantitative reverse-transcription PCR analysis of 96 target genes. Haptoglobin concentrations in plasma were greater for LPS treatment (control = 0.24 ± 0.07, LPS = 0.89 ± 0.06 optical density), but tumor necrosis factor-α concentrations were similar (control = 0.67 ± 0.11, LPS = 0.46 ± 0.11 ng/mL) between treatments. Lipopolysaccharide reduced milk yield after treatment (control = 34.3 ± 1.5, LPS = 29.4 ± 1.6 kg/d), whereas somatic cell count (log) was greater in LPS-treated cows until 34 h after infusions (control = 2.3 ± 0.1, LPS = 3.3 ± 0.1 cells/mL of milk). Rumen-reticular temperature of LPS cows was elevated between 5 and 10 h after each infusion compared with control cows (control = 39.5 ± 0.1, LPS = 40.1 ± 0.1°C). Progesterone concentration after AI was unaffected by treatment or pregnancy status as well as corpus luteum diameter and conceptus length on d 15. Lipopolysaccharide treatment altered the expression of 13 key genes in the endometrium (mostly upregulated), whereas another 17 tended to be modulated. Modified gene expression included genes related to immune response (PTX3 = 2.34-fold increase; IL6 = 3.42-fold increase; and TCN1 = 2.52-fold increase), adhesion molecules (CADM3 = 1.93-fold increase; MMP19 = 1.49-fold increase; EMMPRIN = 1.20-fold increase; SELL = 1.91-fold increase), Wnt signaling pathway (WNT2, FZD4, and FZD7, all <1.5-fold increase), and interferon-stimulated genes (BMP15 = 0.27-fold decrease; ISG15 = 2.17-fold increase, and MX2 = 2.23-fold increase). In summary, intramammary infusions of LPS were able to trigger an inflammatory response with no effect on corpus luteum diameter and concentration of progesterone in plasma. However, a limited but important set of modulations in the endometrium gene expression at d 15 of gestation was found.

Dermatite alérgica à picada de Culicoides em muar: relato de caso / Allergic dermatitis due to bite of Culicoides in muar: case report

A dermatite alérgica à picada de ectoparasitos é uma enfermidade alergoparasitária bastante comum entre animais domésticos, sendo relatada principalmente em pequenos ruminantes e em animais de companhia. Contudo, a doença é pouco diagnosticada na clínica de equídeos devido a similaridades nosológicas com outras dermatopatias. Objetivou-se, com este relato de caso, descrever a síndrome clínica, o plano diagnóstico e a conduta terapêutica de um muar acometido por essa enfermidade. Atendeu-se, no Hospital Veterinário da Universidade Federal Rural do Pernambuco, uma mula de oito anos de idade, que apresentava lesões cutâneas pápulo-crostosas e pruriginosas com evolução clínica de dois anos. Em três situações anteriores, a doença havia sido tratada como dermatite fúngica por outros médicos veterinários. Para o diagnóstico, foram solicitados exame citopatológico e parasitológico de pele, cultivo bacteriológico e fúngico, análise histopatológica e hemograma. Os exames demonstraram uma dermatite superficial perivascular eosinofílica crônica, sendo indicada a terapia tópica com dimetilsufóxido, sulfadiazina, ureia e vitamina A. O protocolo terapêutico mostrou-se satisfatório, permitindo completa remissão do quadro clínico. Este trabalho relatou achados clínicos e patológicos da dermatite alérgica à picada de Culicoides spp. em muar, além de alertar sobre a importância de exames complementares para a realização do diagnóstico diferencial e para o direcionamento terapêutico adequado.(AU)

Allergic dermatitis to ectoparasite bites is a common parasitic disease among domestic animals, being reported mainly in small ruminants and companion animals. However, the disease is poorly diagnosed in equine clinics due to nosological similarities with other skin diseases. The aim of this case report was to describe the clinical syndrome, the diagnostic plan and the therapeutic management of a mule affected by this disease. An 8-year-old mule was observed at Universidade Federal Rural de Pernambuco, presenting papular-crusted and pruritic cutaneous lesions with clinical evolution of two years. In three previous situations, the disease had been treated as fungal dermatitis by other veterinarians. For the diagnosis, cytopathological and parasitological examination of the skin, bacteriological and fungal culture, histopathological analysis and blood count were performed. The exams showed a chronic eosinophilic perivascular superficial dermatitis. A topical therapy with dimethyl sulfoxide, sulfadiazine, urea, and vitamin A was indicated. The therapeutic protocol was satisfactory, allowing complete remission of the clinical condition. This work reported clinical and pathological findings of allergic dermatitis to the bites of Culicoides spp. in muar, in addition to alerting about the importance of complementary examinations for the accomplishment of the differential diagnosis and adequate therapeutic orientation.(AU)

Integrating an automated activity monitor into an artificial insemination program and the associated risk factors affecting reproductive performance of dairy cows.

The aim of this study was to compare 2 reproductive programs for the management of first postpartum artificial insemination (AI) based on activity monitors and timed AI, as well as to determine the effect of health-related factors on detection and expression of estrus. Lactating Holstein cows (n = 918) from 2 commercial farms were enrolled. Estrous cycles of all cows were presynchronized with 2 injections of PGF2α administered 2 wk apart. Treatments were (1) first insemination performed by timed AI (TAI) and (2) first insemination based upon the detection of estrus by activity monitors (ACT; Heatime, SCR Engineering, Netanya, Israel) after the presynchronization, whereas cows not inseminated by the detection of estrus were enrolled in the Ovsynch protocol. Body condition score (BCS; scale 1 to 5), hock score (scale: 1 to 4), gait score (scale: 1 to 4), and corpus luteum presence detected by ovarian ultrasonography were recorded twice during the presynchronization. On the ACT treatment, 50.5% of cows were inseminated based on detected estrus, whereas 83.2% of the cows on the TAI treatment were inseminated appropriately after the timed AI protocol. Pregnancy per AI did not differ by treatment (30.8 vs. 33.5% for ACT and TAI, respectively). Success of pregnancy was affected by parity, cyclicity, BCS, milk production, and a tendency for leg health. In addition, treatment × cyclicity and treatment × parity interactions were found to affect pregnancy success, where anovulatory cows and older cows had compromised pregnancy outcomes on the ACT treatment but not on the TAI treatment. Factors affecting pregnancy outcomes varied among farms. Hazard of pregnancy by 300 DIM was affected by farm, parity, BCS, a treatment × cyclicity interaction, and a tendency for an interaction between leg health and farm. Detection of estrus was affected by farm, parity, cyclicity, and leg health, but not BCS or milk production. Expression of estrus was compromised in anovular and older cows, and by the timing of the estrus event, but not by gait score, BCS, or milk production. Increased duration of estrus, but not intensity of estrus, improved pregnancy per AI. In conclusion, using an automated activity monitor for the detection of estrus within a Presynch-Ovsynch program resulted in similar pregnancy per AI and days open compared with a reproduction program that was strictly based on timed AI for first postpartum AI. In contrast, notable variations in reproductive outcomes were detected between farms, suggesting that the use of automated activity monitors is prone to individual farm management.

Diagnosis of uterine and vaginal disorders by different methodologies is affected by concentration of estradiol in plasma from lactating Holstein cows.

The relationship between plasma estradiol concentration at time of examination and prevalence of uterine disorders, agreement among methods, and associations of diagnosis with pregnancy hazard and milk yield was studied in 268 Holstein cows examined at 30±3 (exam 1) and 44±3 d in milk (DIM; exam 2). Purulent vaginal discharge was sampled using 2 methods: gloved hand and Metricheck (Simcro, Hamilton, New Zealand; PVD; score ≥3). Percentage of polymorphonuclear leukocytes was determined by endometrial cytology (CYTO; exam 1: ≥18%, exam 2: ≥10%); diameter of uterine horns (UTH; >20 mm), diameter of the inner layer of the cervix (CVX; >20.5 mm), presence of fluid in the uterine lumen (FL), and ovarian structures were evaluated by ultrasonography. A blood sample was collected at each exam for estradiol analysis. Prevalence at exams 1 and 2 was, respectively, 14.2 and 18.5% (PVD), 21.4 and 10.1% (FL), and 40.6 and 50.2% (CYTO). Prevalence of PVD at exam 1 was greater among cows with estradiol ≥2 pg/mL (19.4 vs. 8.2%). Agreement of all methods with CYTO was poor, the greatest being between CYTO and FL (exam 1; kappa=0.19). Agreement between CYTO and PVD, and between CYTO and FL (exam 1; kappa=0.15 and 0.35, respectively) was higher among cows with estradiol ≥2 pg/mL. Likelihood of PVD at exam 1 was greater if cows were positive for CVX [odds ratio (OR)=3.0], FL (OR=2.6) or had estradiol ≥2 pg/mL (OR=2.7). Likelihood of CYTO increased with dystocia (OR=2.3) and FL (OR=2.5). Estradiol did not influence diagnosis at exam 2. Positive FL or CYTO at exam 1 was associated with reductions in milk yield of 59 to 180 kg by 45 DIM. Pregnancy hazard until 250 DIM was reduced by CYTO at exam 1 (hazard ratio=0.74) and by PVD (hazard ratio=0.68) at exam 2. However, FL and CYTO reduced pregnancy hazard only when estradiol was ≥2 pg/mL (exam 1), whereas PVD reduced pregnancy hazard when diagnosed at exam 2 with estradiol <2 pg/mL. Overall, agreement was poor and effects of positive diagnosis differed according to method and DIM at exam. Estradiol concentration influenced prevalence, agreement, likelihood of positive diagnosis, and its effects on days to pregnancy.

Expression of estrus modifies the gene expression profile in reproductive tissues on Day 19 of gestation in beef cows.

The aim of this study was to test the effect of expression of estrus at artificial insemination (AI) on endometrium, conceptus, and CL gene expression of beef cows. Thirty-six multiparous nonlactating Nelore cows were enrolled on an estradiol- and progesterone (P4)-based timed AI protocol (AI = Day 0) and then slaughtered for the endometrium, CL, and conceptus collection on Day 19. The animals were retrospectively grouped on the basis of cows that (1) showed signs of estrus near AI (n = 19; estrus) and (2) did not show any signs of estrus (n = 17; nonestrus). Body condition score, blood sampling, and ultrasound examination were performed on Days 0, 7, and 18 of the experiment followed by messenger RNA extraction and quantitative reverse transcription polymerase chain reaction analysis of 58 target genes. Data were checked for normality and analyzed by ANOVA for repeated measures using proc GLM, MIXED, and UNIVARIATE of SAS. Only pregnant cows were included in the analyses (n = 12; nonestrus, n = 11). Estrous expression had no correlation with parameters such as body condition score, preovulatory follicle and CL diameter, P4 concentration in plasma on Days 7 and 18 after AI, and interferon-tau concentration in the uterine flushing (P > 0.15); however, a significant increase was observed in conceptus size from cows that expressed estrus (P = 0.02; 38.3 ± 2.8 vs. 28.2 ± 2.9 mm). The majority of transcripts affected by estrous expression in the endometrium belong to the immune system and adhesion molecule family (MX1, MX2, MYL12A, MMP19, CXCL10, IGLL1, and SLPI; P ≤ 0.05), as well as those related with prostaglandin synthesis (OTR and COX-2; P ≤ 0.05). Genes related to apoptosis, P4 synthesis, and prostaglandin receptor were downregulated (CYP11A, BAX, and FPr; P < 0.05) in the CL tissue of cows that expressed estrus. In addition, four genes were identified as differentially expressed in the 19-day-old conceptus from cows that expressed estrus (ISG15, PLAU, BMP15, and EEF1A1; P < 0.05). There was also a significant effect of Day 7 concentration of P4 mainly affecting the immune system, adhesion molecules, and wnt signaling pathway of the endometrium (IGLL1, MX2, SLPI, TRD, APC, WNT2, GLYCAM1, and MYL12A; P < 0.05). A significant interaction between estrous expression and P4 concentration on Day 7 was more pronounced in immune system genes (MX1, MX2, TRD, SLPI, and IGLL1; P < 0.05). This study reported that estrous expression at the time of AI favorably altered the gene expression profile in reproductive tissues during the preimplantation phase toward a more receptive state to the elongating conceptus. These effects seem to be more evident in the endometrium during the time of dynamic remodeling for embryo implantation.

Intercorrência entre leucose enzoótica e tuberculose em bovinos leiteiros do estado de Pernambuco / Assessment of intercurrence of enzootic leucosis and bovine tuberculosis in dairy cows in the state of Pernambuco, Brazil

Objetivou-se com este estudo avaliar a intercorrência entre leucose enzoótica e tuberculose em rebanhos bovinos leiteiros em oito municípios do Estado de Pernambuco, pelo estabelecimento da prevalência de bovinos reagentes às provas diagnósticas específicas. Foram analisados sorologicamente 662 animais, pela técnica de imunodifusão dupla em gel de agarose e 612 foram avaliados imunoalergicamente, por meio do teste de tuberculinização. Nos 15 rebanhos examinados, as prevalências de bovinos que apresentaram positividade aos testes IDGA e tuberculina foram 32,2% (213/662) e 14,1% (86/612), respectivamente. Os resultados obtidos nesta pesquisa permitiram concluir que as doenças estudadas encontram-se amplamente disseminadas na população avaliada, com crescimento em níveis significativos da leucose, e as infecções pelo vírus da leucose bovina (VLB) e Mycobacterium bovis encontram-se ativas e em expansão, com risco do comprometimento da saúde dos rebanhos bovinos e, pelo caráter zoonótico da tuberculose bovina, das pessoas que lidam com os bovinos.

We carried out this study to evaluate the intercurrence of tuberculosis and enzootic leukosis in dairy cattle herds in 8 districts of the State of Pernambuco, Brazil, by establishing the prevalence of bovines reagent to specific diagnostic tests, and to also verify the correlation between enzootic leucosis, cattle tuberculosis, the leukocytes count and lymphocytes count among the cattle studied. A total of 662 animals were tested serologically and hematologically, by the techniques of double immunodiffusion in agarose gel and the white blood cell count with the total and differential leukocyte count, respectively, and 612 were evaluated immunoallergically by the tuberculin test. In the 15 herds examined, the prevalence of cattle that were positive to the AGID and tuberculin tests were 32.2% (213/662) and 14.1% (86/612), respectively. Analyzing the correlation between the variables total leukocytes and lymphocytes, we found that total leukocyte is highly related to the variable lymphocytes (72%). The results in this study indicated that the diseases studied are widespread in the population studied, with significant levels of growth in leukemia, and infections of bovine leukosis virus (VLB) and Mycobacterium bovis are active and growing, with risk of impairment of the health of cattle herds, and, due to the zoonotic nature of bovine tuberculosis, of the health of people who work with cattle.