Aborto infeccioso por Neospora spp. em equino ˗ relato de caso / [Neospora spp infectious abortion in the horse ˗ case report]

O abortamento na espécie equina é responsável por grandes perdas econômicas e, entre as possíveis causas, está a neosporose, uma enfermidade que nem sempre é investigada como provável diagnóstico. Assim, o objetivo deste trabalho foi relatar um caso de aborto equino aos 129 dias de gestação, resultante da infecção por Neospora spp.. Amostras sanguíneas da égua e do feto abortado foram enviadas para um laboratório especializado. À necrópsia, constatou-se edema gelatinoso e hemorrágico em subcutâneo, fígado ictérico e friável com autólise de alguns órgãos, como baço, rins e glândulas adrenais. Fragmentos dos órgãos coletados na necrópsia foram submetidos à histopatologia e à pesquisa de agentes infecciosos, em que se identificou hepatite e epicardite não purulentas e onfalite purulenta, bem como exame positivo para Neospora spp. pela técnica de reação em cadeia da polimerase (PCR) convencional e Nested. O soro sanguíneo obtido da égua foi submetido à pesquisa de Neospora spp. pela técnica de imunofluorescência indireta, de herpesvírus por soroneutralização em cultura de células e de Leptospira sp. por PCR, todas com resultados negativos. Dessa forma, é importante considerar a neosporose como diagnóstico diferencial em casos de abortamento e natimortalidade, uma vez que a presença de cães nas propriedades é extremamente comum, representando uma importante fonte de infecção.(AU)

Abortion in the equine species is responsible for major economic losses, and among the possible causes is neosporosis, a disease that is not always investigated as a probable diagnosis. Thus, the objective of this study was to report an abortion at 129 days of gestation resulting from Neospora spp. Blood samples from the mare and the aborted fetus were sent to a specialized laboratory. Under necropsy, gelatinous and hemorrhagic edema was detected in subcutaneous tissue, friable and jaundiced liver with autolysis in spleen, kidneys and adrenal glands. Fragments of the organs collected at necropsy were submitted to histopathology and infectious agent tests, which identified non-purulent hepatitis and epicarditis and purulent omphalitis. Also, a positive result for Neospora spp. by the conventional and nested Polymerase Chain Reaction (PCR) technique. Blood serum obtained from the mare was subjected to analyses to Neospora spp. by indirect immunofluorescence technique, herpesvirus by serum neutralization in cell culture and Leptospira sp. by PCR, all with negative results. Thus, it is important to consider neosporosis as a differential diagnosis in cases of abortion and stillbirth, since the presence of dogs in the properties is extremely common and represent an important source of infection.(AU)

Uterine cervix as a fundamental part of the pathogenesis of pregnancy loss associated with ascending placentitis in mares.

Anatomical and molecular changes in the cervical barrier in women are a fundamental part of the pathogenesis of pregnancy loss associated with chorioamnionitis. However, there is little information regarding changes in the cervix associated with ascending infection in pregnant mares. To better characterize morphological and molecular changes in the cervix during placentitis, we examined full thickness histology and mRNA expression for a number of inflammatory and endocrine factors in the mucosa and stroma of the cervix of mares (n = 5) after experimental induction of placentitis via transcervical inoculation with Streptococcus equi ssp zooepidemicus at approximately 290d of gestation. Gestationally age-matched mares (n = 4) served as controls. Target transcripts included steroid receptors (PGR, ESR1 and 2), OXTR, prostaglandins synthases and receptors (PTGS1, PTGS2, PGES, PGFS, PTGER2 and PTGER4), cytokines (IL1b, IL6, CLCX8, IL10 and TNFα) and acute phase proteins (SAA). Histologically, a marked modification in the cervical epithelia and stroma was characterizing cervicitis. Additionally, the mRNA expression of IL1ß, IL6, CXCL8, SAA and PTGS2 was greater (P < 0.05) in both mucosa and stroma of the inoculated mares; whereas TNFα, IL10 and PGES were upregulated (P < 0.05) only in the cervical mucosa. Progesterone receptor, ESR1 and PTGER4 were upregulated in the cervical stroma of placentitis mares. In conclusion, the cervical response to placentitis was characterized by an upregulation of inflammatory cytokines that was accompanied by induction of PTGS2 and PGES. Further, receptors known to be associated with relaxation of the cervix in other species (ESR1 and PTGER4) were upregulated in the cervical stroma of placentitis mares. These findings indicate that the cervix is not only a physical barrier but that it has an active role in the pathogenesis of ascending placentitis.

The effect of flunixin meglumine, firocoxib and meloxicam on the uterine mobility of equine embryos.

Embryo mobility occurs as a result of prostaglandin production by the embryo and endometrium, promoting uterine smooth muscle contractions, which propels the embryonic vesicle through the lumen. Non-steroidal anti-inflammatory drugs (NSAIDs), as flunixin meglumine, are routinely used in equine medicine and can alter the conceptus mobility if applied in early pregnancy, which may impair maternal recognition of pregnancy. The objective of this study was to evaluate and compare the effect of flunixin meglumine (FM; 1.1 mg/kg IV), firocoxib (FIRO; 0.2 mg/kg PO), and meloxicam (ML; 0.6 mg/kg, IV), on the embryo mobility. Thirty mares were divided into three groups (n = 10 per treatment). After the pregnancy diagnosis on day 12 after ovulation, the embryo mobility was evaluated by transrectal ultrasonography every 5 min for 1 h in order to visualize the location of the embryo. In all mares, three evaluations were performed: immediately before treatment (pre-treatment), after NSAID administration and 24 h after treatment. In group FM, embryo mobility decreased, from 5.8 ±â€¯0.3 movements/hour (m/h) to 2.3 ±â€¯0.5 m/h (p < 0.05) and, after 24 h the values were similar to the pre-treatment evaluation (5.9 ±â€¯0.2 m/h). Likewise, ML treatment caused a decrease of embryo movements, from 5.9 ±â€¯0.3 to 1.9 ±â€¯0.3 m/h (p < 0.05), 24 h after treatment values were 5.7 ±â€¯0.4 m/h. Treatment with FIRO did not interfere with embryo mobility (5.7 ±â€¯0.4; 5.8 ±â€¯0.3 and 5.6 ±â€¯0.3 embryo movements in the first, second and third evaluation, respectively). In conclusion, FIRO was the only NSAID that did not alter the embryo mobility and may be the safest NSAID for use in early pregnant mares.

Mammary botryomycosis: two concomitant cases in mares: case report / Botriomicose mamária: dois casos concomitantes em éguas: relato de caso

Mammary disorders in mares are rare and usually only one animal per paddock is affected. In this report, two mares with 7 and 9 years old, were concomitantly diagnosis of chronic pyogranulomatous mastitis, associated with the Splendore-Hoeppli reaction, indicative of botryomycosis a rare, chronic suppurative disease with microabscess formation, characterized by in vivo formation of eosinophilic materials around microorganisms or biologically inert material. Various bacteria can cause botryomycosis in horses, and the most frequently isolated one is Staphylococcussp., particularly S. aureus. This report confirms the role of Staphylococcus sp.; however, specifically S. hyicus and S. cohnii that prior to the current report, had not been associated with cases of botryomycosis.(AU)

Enfermidades mamárias em éguas são raras e, normalmente, apenas um animal é afetado. Neste relato, duas éguas, de sete e nove anos, foram diagnosticadas concomitantemente com mastite crônica piogranulomatosa, associada com reação de Splendore - Hoeppli, indicativa de botriomicose, uma doença crônica supurativa rara, com formação de microabscessos caracterizados pela presença de material eosinofílico em torno dos microrganismos ou de material biologicamente inerte. Várias bactérias podem causar botriomicose em cavalos, sendo Staphylococcus sp., particularmente S. aureus, as mais frequentemente isoladas. Este relato confirma o papel do Staphylococcus sp, no entanto este é o primeiro relato em que S. hyicus e S. cohnii foram identificados em lesões relacionadas à botriomicose.(AU)

Compartment syndrome after South American rattlesnake (Crotalus durissus terrificus) envenomation.

CONTEXT: In order to report the outcome of a patient who developed compartment syndrome after South American rattlesnake (Crotalus durissus terrificus) envenomation, confirmed by subfascial pressure measurement and magnetic resonance imaging (MRI). CASE DETAILS: A 63-year-old male was admitted 1 h after being bitten on the right elbow by a "large" snake, which was not brought for identification. Physical and laboratory features upon admission revealed two fang marks, local tense swelling, paresthesia, intense local pain, hypertension, coagulopathy, and CK = 1530 U/L (RV < 170 U/L). The case was initially treated with bothropic antivenom (80 mL, intravenously), with no improvement. Evolution within 13-14 h post-bite revealed generalized myalgia, muscle weakness, palpebral ptosis, and severe rhabdomyolysis (CK = 126,160 U/L) compatible with envenoming by C. d. terrificus. The patient was then treated with crotalic antivenom (200 mL, intravenously), fluid replacement, and urine alkalinization. Twenty-four-hour post-bite MRI showed marked muscular edema in the anterior compartment of the right forearm, with a high subfascial pressure (40 mmHg) being detected 1 h later. ELISA of a blood sample obtained upon admission, before antivenom infusion, revealed a high serum concentration of C. d. terrificus venom. No fasciotomy was performed and the patient was discharged seven days later without sequelae. CONCLUSION: Snakebite by C. d. terrificus with subfascial venom injection may lead to increased intracompartmental pressure.

Detection of periodontal pathogens in newborns and children with mixed dentition.

We report the age-related prevalence of red complex periodontal pathogens, Porphyromonas gingivalis, Treponema denticola, and Tannerella forsythia, along with four strains of orange complex pathogens. The bacteria present in samples isolated from tongue, cheek, and subgingival sulcus in edentulous newborns and children with mixed dentition were monitored by polymerase chain reaction (PCR). P. gingivalis was not detected in any site of any subject in the two groups tested. However, T. denticola was not only found in the 6-13 years age group, but also in edentulous newborns at a relatively high prevalence, indicating non-dentition-related colonization by T. denticola. Campylobacter rectus, Prevotella intermedia, T. forsythia, Eikenella corrodens, and Parvimonas micra were found in the oral cavity of most subjects belonging to the 6-13 years age group compared to newborns. This suggested a pronounced association between these colonizing bacteria and the presence of teeth. There was also a strong relation between T. denticola and T. forsythia for their prevalence in the subgingival sulcus of the 6-13 years age group (p < 0.0001), but not in the other sites tested, suggesting that the colonization of dentition-related T. forsythia may be associated with the increased prevalence of non-dentition-related T. denticola in the subgingival sulcus. Overall, these results suggest that dentition is a key determinant of bacterial colonization, especially orange complex bacteria and the red complex bacterium T. forsythia.

Viability and cell cycle analysis of equine fibroblasts cultured in vitro.

This experiment aimed to study equine fibroblasts in culture analyzing and the cell cycle and viability of cells pre- and post-freezing. Skin fragments were obtained from 6 horses and cultured in DMEM high glucose + 10% FCS in 5% CO(2) until the beginning of confluence. Two passages were performed before freezing. Cells subjected to serum starvation (0.5% FCS) were analyzed for viability and cell cycle at 24, 48, 72, 96, 120, 144 and 168 h of culture. For the confluent groups, cells were analyzed at the moment they achieved confluence. Cellular viability was assisted with Hoescht 33342 and propidium iodide. The analysis of apoptosis/necrosis and cell cycle was performed using a flow cytometer (FACS Calibur BD((R))) after staining the cells with annexin V and propidium iodide. Both optical microscopy and flow cytometry confirmed that cellular viability was similar for serum starvation and confluent groups (average 84%). Similarly, both methods were efficient to synchronize the cell cycle before freezing. However, after thawing, serum starvation, for more than 24 h, was superior to culture for synchronizing cells in G0/G1 (69% x 90%). The results of this experiment indicate that equine fibroblasts can be efficiently cultured after thawing.

Use of a Piezo drill for intracytoplasmic sperm injection into cattle oocytes activated with ionomycin associated with roscovitine.

Intracytoplasmic sperm injection (ICSI) consists of the introduction, by micromanipulation, of a single sperm into the cytoplasm of a mature egg. This technique is particularly advantageous when only a few sperm are available for fertilization, representing an important tool in preserving genetic material, especially from poorly fertile males. The results from ICSI in cattle are very often unsatisfactory and difficult to reproduce. Thus, the goal of this study was to evaluate the effect of the use of a Piezo drill (PD) and oocyte activation with ionomycin + roscovitine (I + R) during ICSI in cattle oocytes. After in vitro maturation (24 h), cumulus complex oocytes were divided into four groups: G1 - the ICSI was performed without the use of a PD and the oocyte was activated with I + R; G2 - the ICSI was performed with the use of the PD and activation with I + R; G3 - the ICSI was performed with the use of the PD, but without activation and G4 - parthenogenetic control, treated with I + R, but without sperm injection. The presumptive zygotes were cultured for 7 days and evaluated on day 3 for cleavage rate and on day 7 for blastocyst formation. Embryo production by standard in vitro fertilization in the laboratory was 78% for cleavage (117/150) and 35% for blastocyst formation (41/150). The cleavage rates obtained in G1, G2 and G4 were similar (66.7%, 71.6% and 66.3%, respectively), demonstrating the beneficial effect of oocyte activation. However, in G3, despite the presence of the sperm and the electric stimulation of a PD, the cleavage rates were significantly lower (17.5%) compared with the groups that used chemical activation, even in the absence of sperm (G4). Despite the beneficial effects of activation, this stimulus alone, or in the absence of the PD, was not sufficient for adequate morulae formation (13.4%, 37.9%, 0.0% and 13.5% for G1, G2, G3 and G4, respectively). Only in G2, when the PD was used followed by artificial activation, blastocysts were obtained (14.7%). These results indicate that cattle oocytes must be activated after ICSI to produce viable embryos.

Ultrastructural characteristics of non-matured and in vitro matured oocytes collected from pre-pubertal and adult domestic cat ovaries.

The present study describes the ultrastructural characteristics of cat oocytes before maturation and after 12- and 24-h in vitro maturation (IVM). Oocytes were recovered from pre-pubertal and adult queen ovaries after ovariohysterectomy and a proportion were stored in glutaraldehyde at 4 degrees C until examination by transmission electronic microscopy (TEM). Those selected for maturation were cultured before TEM in DMEM for 12 and 24 h at 38 degrees C in a humidified environment of 5% O(2), 5% CO(2) and 90% N(2). Specimens were divided into six groups: non-matured oocytes from pre-pubertal queens (PP0), non-matured oocytes from adult queens (A0), 12-h in vitro matured oocytes from pre-pubertal queens (PP12), 12-h in vitro matured oocytes from adult queens (A12), 24-h in vitro matured oocytes from pre-pubertal queens (PP24) and 24-h in vitro matured oocytes from adult queens (A24). Across the treatment groups, it was possible to observe differences in the thickness of the perivitelline space, the penetration of cumulus cell projections forming a junctional complex, distribution and density of small vesicles, lipid droplets, microvilli, mitochondria and cortical granules and variable degrees of development of Golgi complexes. These findings demonstrated that ultrastructural analysis of oocytes matured in vitro is a valuable tool to evaluate oocyte cytoplasmic maturation and that this IVM protocol was efficient in inducing gradual morphological changes necessary for cytoplasmic maturation of pre-pubertal and adult cat oocytes.

Aspiration of equine oocytes from immature follicles after treatment with equine pituitary extract (EPE) alone or in combination with hCG.

This study examined the effect of treating mares with equine pituitary extract (EPE) alone or in combination with hCG on the recovery rate of immature follicles by transvaginal follicular aspiration (ovum pick-up; OPU). Ten normally cycling crossbred mares aged 3-15 years and weighing 350-400 kg were subjected to each of three treatments in a random sequence with each exposure to a new treatment separated by a rest cycle during which a spontaneous ovulation occurred. The treatments were (1) superovulated with 25mg EPE and treated with 2500 IU hCG, (2) superovulation with 25mg EPE, and (3) control (no exogenous treatment). Treatments 7 days after spontaneous ovulation; and all the follicles >10mm were aspirated 24h after the largest follicle achieved a diameter of 27-30 mm for control group, and most follicles reached 22-27 mm for the EPE alone treatment. To the group EPE+hCG, when the follicles reached 22-27 mm, hCG was administered, 24h before OPU. Superovulation increased the number of follicles available for aspiration. The total number of follicles available for aspiration was 61 in the EPE/hCG group, 63 in the EPE group and 42 in the control. The proportion of follicles aspirated varied from 63.5% to 73.8%. Oocyte recovery rate ranged from 15.0% to 16.7% and the proportion of mares that yielded at least one oocyte was 70% (7/10) in the EPE/hCG, 60% (6/10) in the EPE alone and 50% (5/10) in control group. The EPE/hCG treatment had a higher proportion of follicles with expanded granulose cells (64.4%) than the control (3.3%; p<0.05) and the EPE treatment (25.0%). The intervals from spontaneous ovulation to aspiration were similar for all treatments (11-12 days). However, superovulatory treatment significantly increased the aspiration to ovulation interval from 15+/-4 days for control to 27+/-15 days for EPE (p<0.05) and to 23+/-13 days for EPE/hCG treatment with commensurate increases in the time between spontaneous ovulations.