Sheep as a new experimental host for Babesia divergens

Babesia divergens was cultivated in sheep erythrocytes in RPMI 1640 supplemented with 10% Fetal Calf Serum (FCS) or sheep serum. In vitro cultures in sheep red blood cells were initiated with human erythrocytes infected in vitro with B. divergens Rouen 1987 or with gerbil blood infected with several isolates from bovine origin. After the first subcultures on sheep erythrocytes, a ten-fold multiplication of the parasites was obtained within 48 h. Erythrocytes from three splenectomized sheep were infected in vitro with B. divergens; when parasitaemia reached 10%, the animals were inoculated with homologous parasitized erythrocytes. All sheep expressed hyperthermia with a peak between the 6th and the 9th day post-infection (p-i) and a transitory parasitaemia 10 days p-i. In vitro primary cultures were performed on two of these sheep, demonstrating the parasite persistence at very low parasitaemia in the infected animals. Splenectomized sheep can be used as a new model for B. divergens chronic infection.     

Isolation and characterization of Babesia pecorum sp. nov. from farmed red deer (Cervus elaphus)

The diversity of Babesia species infecting cervids in parts of central and southern Spain was analyzed by collecting blood from farmed red deer (Cervus elaphus). Babesia sp. was isolated in vitro from two red deer herds in Cádiz and Ciudad Real. The number of Babesia sp. carriers differed between the two herds: 36/77 in Cádiz and 1/35 in Ciudad Real. Hyalomma lusitanicum was the most prevalent tick species identified on the Cádiz farm vegetation and on sampled animals, and is therefore a candidate vector. The molecular characteristics of 21 isolates were determined by complete (8 isolates) or partial (13 isolates) 18S rRNA gene sequencing. The sequences were highly similar (over 99.4% identity) and 6 sequence types were identified at the level of one herd only, demonstrating a rather high genetic diversity. They formed a monophyletic clade, and members of the three main sequence types shared a similar morphology and the same erythrocyte susceptibility pattern. This clade also included Babesia sp. Xinjiang isolated from sheep in China and Babesia sp. identified in giraffe in South Africa, with identities higher than 98.3% and statistically relevant phylogenetic support. None of the biological properties analyzed for both Babesia from red deer and Babesia sp. Xinjiang allowed their differentiation (ability to develop in vitro in erythrocytes from cattle and sheep, as well as in erythrocytes from different cervids, unsuccessful infection of calves). We propose the Babesia isolated from red deer as a new species named B. pecorum. Whether Babesia sp. Xinjiang and the Babesia characterized in South Africa belong to the same species is debated.

Electronic supplementary material

The online version of this article (doi:10.1186/s13567-014-0078-7) contains supplementary material, which is available to authorized users.     

H3S10 Phosphorylation Marks Constitutive Heterochromatin During Interphase in Early Mouse Embryos Until the 4-Cell Stage

Phosphorylation of histone H3 at Ser10 (H3S10P) has been linked to a variety of cellular processes, such as chromosome condensation and gene activation/silencing. Remarkably, in mammalian somatic cells, H3S10P initiates in the pericentromeric heterochromatin during the late G2 phase, and phosphorylation spreads throughout the chromosomes arms in prophase, being maintained until the onset of anaphase when it gets dephosphorylated. Considerable studies have been carried out about H3S10P in different organisms; however, there is little information about this histone modification in mammalian embryos. We hypothesized that this epigenetic modification could also be a marker of pericentromeric heterochromatin in preimplantation embryos. We therefore followed the H3S10P distribution pattern in the G1/S and G2 phases through the entire preimplantation development in in vivo mouse embryos. We paid special attention to its localization relative to another pericentromeric heterochromatin marker, HP1β and performed immunoFISH using specific pericentromeric heterochromatin probes. Our results indicate that H3S10P presents a remarkable distribution pattern in preimplantation mouse embryos until the 4-cell stage and is a better marker of pericentromeric heterochromatin than HP1β. After the 8-cell stage, H3S10P kinetic is more similar to the somatic one, initiating during G2 in chromocenters and disappearing upon telophase. Based on these findings, we believe that H3S10P is a good marker of pericentromeric heterochromatin, especially in the late 1- and 2-cell stages as it labels both parental genomes and that it can be used to further investigate epigenetic regulation and heterochromatin mechanisms in early preimplantation embryos.     

Risk Factors for Incisional Complications after Exploratory Celiotomy in Horses: Do Skin Staples Increase the Risk?

Objective: To assess risk factors for celiotomy incisional infection in horses, especially the use of staples for skin closure. Study Design: Case series. Animals: Horses (n=356) that had 1 exploratory celiotomy for colic and survived >2 weeks after surgery between March 1, 2004 and December 31, 2007. Methods: Incisions were classified as “normal” (no complication, only edema, serous drainage lasting <24 hours) or as “surgical site infection (SSI)” (persistent serosanguinous drainage or purulent drainage with or without positive bacterial culture). All possible risk factors, including method of skin closure (monofilament sutures or staples), were statistically analyzed using univariate and multivariate logistic regression. Results: Of 356 horses, 303 (85%) had normal wound healing and 53 (15%) developed a SSI (purulent: 48 [14%]; persistent serosanguinous: 5 [1%]). Bacterial cultures were positive in 33 of 40 cases. Factors significantly associated with SSI in the multivariate analysis were: use of staples for skin closure (odds ratio [OR] 3.85, P<.001) and surgical site closure by a 1st or 2nd year resident (OR 2.20, P=.016). Lavage of the linea alba with sterile saline solution after closure was a protective factor (OR 0.38, P=.004). Conclusion: Use of staples for skin closure and less experienced surgeons closing the abdomen are risk factors for incisional infection. Incisional lavage after linea alba closure was a protective factor. Clinical Relevance: Despite their ease and speed of application, skin staples can lead to an increase in celiotomy wound complications in horses.     

The fungal T-2 toxin alters the activation of primary macrophages induced by TLR-agonists resulting in a decrease of the inflammatory response in the pig

T-2 toxin is known to be one of the most toxic trichothecene mycotoxins. Exposure to T-2 toxin induces many hematologic and immunotoxic disorders and is involved in immuno-modulation of the innate immune response. The objective of this work was to evaluate the effects of T-2 toxin on the activation of macrophages by different agonists of Toll-like receptors (TLR) using an in vitro model of primary porcine alveolar macrophages (PAM). Cytotoxic effects of T-2 toxin on PAM were first evaluated. An IC₅₀ of 19.47 ± 0.9753 nM was determined for the cytotoxicity of T-2 toxin. A working concentration of 3 nM of T-2 toxin was chosen to test the effect of T-2 toxin on TLR activation; this dose was not cytotoxic and did not induce apoptosis as demonstrated by Annexin/PI staining. A pre-exposure of macrophages to 3 nM of T-2 toxin decreased the production of inflammatory mediators (IL-1 beta, TNF-alpha, nitric oxide) in response to LPS and FSL1, TLR4 and TLR2/6 agonists respectively. The decrease of the pro-inflammatory response is associated with a decrease of TLR mRNA expression. By contrast, the activation of TLR7 by ssRNA was not modulated by T-2 toxin pre-treatment. In conclusion, our results suggest that ingestion of low concentrations of T-2 toxin affects the TLR activation by decreasing pattern recognition of pathogens and thus interferes with initiation of inflammatory immune response against bacteria and viruses. Consequently, mycotoxins could increase the susceptibility of humans and animals to infectious diseases.     

Analysis of cellular responses to Mycoplasma mycoides subsp. mycoides small colony biotype associated with control of contagious bovine pleuropneumonia

A better understanding of protective immune memory against contagious bovine pleuropneumonia (CBPP) is needed in order to facilitate the development of safer vaccines based on selected components of the pathogen. For this purpose, cells collected from lymph nodes draining the lungs of Mycoplasma mycoides subsp. mycoides small colony biotype (MmmSC)-infected cattle were stimulated with the pathogen in vitro and evaluated concurrently for proliferation (CFSE based method), expression of activation, memory markers and cytokine production. Direct evidence is presented for a major contribution of CD4+ T cells to the vigorous proliferative and T1 biased cytokine recall responses observed in cattle that have recovered from infection but not in animals developing the acute form of the disease. Two different phenotypes of MmmSC-specific memory CD4 were observed based on CD62L expression and proliferative capacities. Furthermore, recall proliferation of B cells also occurred but was strictly dependent on the presence of CD4. The information provided in this study will facilitate the search for MmmSC antigens that have potential for the development of subunit vaccines against CBPP.     

ULTRASONOGRAPHIC FINDINGS IN 13 HORSES WITH LYMPHOMA

Ultrasonography and radiography are commonly used for staging of lymphoma in horses, however there is little published information on imaging characteristics for horses with confirmed disease. The purpose of this retrospective, case series study was to describe ultrasonographic and radiographic findings for a group of horses with a confirmed diagnosis of lymphoma. A total of 13 horses were sampled. Lymphadenopathy (8/13), peritoneal effusion (6/13), splenic (6/13), and hepatic (5/13) lesions were the most frequently identified. The predominant splenic and hepatic ultrasonographic lesions were hypoechoic nodules, organomegaly, and changes in echogenicity. Digestive tract lesions were detected in three horses and these included focal thickening and decreased echogenicity of the small (2/13) and large intestinal (2/13) wall. Thoracic lesions were predominantly pleural effusion (4/13), lymphadenopathy (4/13), and lung parenchymal changes (3/13). Enlarged lymph nodes were detected radiographically (4/13) and/or ultrasonographically (2/13) in the thorax and ultrasonographically in the abdomen (7/13) and in the caudal cervical region (4/13). Findings supported the use of abdominal and thoracic ultrasonography for lymphoma staging in horses. Ultrasound landmarks for localizing cecal and caudal deep cervical lymph nodes were also provided.