荷兰奶业产业链管理及质量控制

在第四届中国国际奶业展览会及高层论坛上,韩国、日本、德国、荷兰奶业交流专场吸引了众多中外奶业界同仁。国外专家关于本国奶牛养殖业、乳品加工业的精彩报告,使与会代表对各国的奶业发展现状和趋势有了全面而深入地了解。本刊编辑部,对各位国外专家的发言进行了整理,现分韩国篇、日本篇、德国篇、荷兰篇四个部分予以刊登,以飨读者,希望对中国建设现代奶业有所借鉴。     

Enhancement of the interferon gamma assay to detect paratuberculosis using interleukin-7 and interleukin-12 potentiation

A limitation to the widespread application of interferon gamma (IFN-γ) response assays has been logistical difficulties, as they must be performed within hours of blood collection. Detection of specific IFN-γ responses to Mycobacterium avium subspecies paratuberculosis (MAP) as part of the cell-mediated immune response of ruminants with Johne's disease could aid in diagnosis and control programs. In this study, a modified protocol was developed in which cultures were supplemented with interleukin (IL)-7, a survival factor required to maintain resting T cells, alone or in combination with IL-12 to potentiate IFN-γ responses and extend blood storage time. The combination of IL-7 and IL-12 was synergistic, giving IFN-γ responses greater than with IL-12 alone, for sheep blood stored up to 2 days. In a cohort of naturally infected sheep, the same number of animals was identified as test positive using the modified assay (performed after 2 days with IL-7/IL-12 supplementation) as the standard IFN-γ assay performed on the day of blood collection. The modified assay offered greatest advantage in the detection of early stages of paratuberculosis infection, for sheep with low grade and paucibacillary lesions, and at early time points post-infection. The potentiation protocol allowed for practical shipment of blood samples from farm to laboratory, extending permissible transit times and applicability of IFN-γ testing to detect Johne's disease.     

Safety and efficacy of an oil-adjuvant vaccine against haemorrhagic septicaemia in buffalo calves: cross-protection between the serotypes B:2,5 and E:2,5.

The safety, efficacy and duration of immunity of an improved oil-adjuvant vaccine against haemorrhagic septicaemia, containing inactivated cells of Pasteurella multocida serotype B:2,5, were tested in young buffalo calves in Pakistan. For safety testing, five buffalo calves were vaccinated intramuscularly with twice the normal dose, and six weeks later with a normal dose. Except for a transient rise in rectal temperature at six hours after the vaccinations, no systemic reactions were observed. The buffaloes remained in good condition and had a normal appetite. No local reactions were observed at the injection site. For efficacy testing two trials were carried out. In the first, buffalo calves were vaccinated intramuscularly either with two doses two-and-a-half months apart, or with a single dose, or left unvaccinated. They were challenged subcutaneously with virulent P multocida after eight, 13 or 15 months. After challenge at eight months the four buffaloes given two doses and the buffalo given one dose were protected, whereas the control animal developed the typical signs of the disease. After the challenges at 13 and 15 months, the vaccinated animals were still protected whereas the control animals died. In the second trial, buffalo calves were vaccinated intramuscularly either with two doses two months apart, or with a single dose at two months or left unvaccinated. The buffaloes were challenged after eight or 14 months. After challenge at eight months the four control animals died, whereas three of the four buffaloes given a single dose were protected. After challenge at 14 months, the three control animals died, whereas four of the five buffaloes given two doses and both the buffaloes given a single dose were protected. To test for cross-protection against the heterologous serotypes E:2,5 and B:3,4, groups of mice were vaccinated once or left unvaccinated. Four weeks later, the vaccinated and control groups were challenged with a dilution series of the different challenge cultures. The vaccine appeared to induce protection against challenge with different strains of serotypes B:2,5 and E:2,5 but not against strains of serotype B:3,4.     

Virulence-associated trimeric autotransporters of Haemophilus parasuis are antigenic proteins expressed in vivo

Glässer’s disease is a re-emerging swine disease characterized by a severe septicaemia. Vaccination has been widely used to control the disease, although there is a lack of extended cross-protection. Trimeric autotransporters, a family of surface exposed proteins implicated in host-pathogen interactions, are good vaccine candidates. Members of this family have been described in Haemophilus parasuis and designated as virulence-associated trimeric autotransporters (VtaA). In this work, we produced 15 recombinant VtaA passenger domains and looked for the presence of antibodies directed against them in immune sera by immunoblotting. After infection with a subclinical dose of H. parasuis Nagasaki, an IgG mediated antibody response against 6 (VtaA1, 5, 6, 8, 9 and 10) of the 13 VtaA of the Nagasaki strain was detected, indicating that they are expressed in vivo. IgA production against VtaA was detected in only one animal. VtaA were more likely to be late antigens when compared to early (Omp P5 and Omp P6) and late (YaeT) defined antigens. Antibody cross-reaction with two orthologs of Nagasaki’s VtaA5 and 6, VtaA15 and 16 of strain HP1319, was also detected. No antibodies against VtaA were detected in the sera of animals immunized with a bacterin of the Nagasaki strain, suggesting poor expression in the in vitro conditions used. Taken together, these results indicate that VtaA are good candidate immunogens that could be used to improve H. parasuis vaccines. However, their capacity to confer protective immunity needs to be further studied.     

Evaluation of Praziquantel effects on Echinostoma paraensei ultrastructure

Echinostomiasis is a food-borne, intestinal, zoonotic, snail-mediated helminthiasis caused by digenean trematodes of the family Echinostomatidae with seven species of the genus Echinostoma infecting humans or domestic and wildlife animals. Echinostoma paraensei is a peristomic 37-collar-spined echinostome belonging to the “revolutum group”.     

Beacon-like immunoreactivity in the hypothalamus of domestic chick

Beacon-immunoreactive (B-ir) fibres and neurons in the hypothalamus of the domestic chick (Gallus domesticus) were studied using an immunohistochemical technique in order to verify the presence and elucidate the pattern of distribution of this novel peptide in an avian brain. B-ir neurons were seen in the n. supraopticus, pars ventralis and pars externus; n. magnocellularis preopticus, pars dorsalis, medialis and ventralis; n. preopticus periventricularis; n. suprachiasmaticus, pars medialis; n. ventrolateralis thalami. Only few B-ir cells were scattered in the most anterior part of the lateral hypothalamic area. B-ir fibres, appearing as thin punctuate structures, were seen mainly along the walls of the third ventricle and in the ventromedial hypothalamus. Labelled fibres and terminals were located in the external and internal zones of the anterior and posterior median eminence. In particular, fibre terminals were seen close to the capillary loops of the hypothalamo-hypophysial portal system. The anatomical data of the present study regarding the distribution of B-ir in the chick hypothalamus suggest that beacon may play a key role in the regulation of the neuroendocrine system by acting as a neuromodulator and/or neurotransmitter.     

A survey of mutations in the Cochliomyia hominivorax (Diptera: Calliphoridae) esterase E3 gene associated with organophosphate resistance and the molecular identification of mutant alleles

Cochliomyia hominivorax (Calliphoridae) is one of the most important myiasis-causing flies and is responsible for severe economic losses to the livestock industry throughout the Neotropical region. In Brazil, C. hominivorax has been controlled mainly with organophosphate (OP) insecticides, although the inappropriate use of these chemicals can result in the selection of resistant flies. Changes in carboxylesterase activity have been associated with OP insecticides in some arthopodan species. In this work, we isolated and characterized part of the E3 gene in C. hominivorax (ChE7), which contained the same substitutions responsible for the acquisition of OP hydrolase activity in Lucilia cuprina (Calliphoridae). Digestion of the polymerase chain reaction products with a restriction enzyme that specifically recognized the mutation site unambiguously differentiated wild and mutated esterase alleles. The PCR-RFLP assay therefore provided a fast, reliable DNA-based method for identifying C. hominivorax individuals with a mutation in the esterase gene. Further bioassays to determine the association of this mutation with OP resistance in C. hominivorax should allow the development of more effective strategies for managing this species.     

Bluetongue in The Netherlands; description of the first clinical cases and differential diagnosis. Common symptoms just a little different and in too many herds

For the first time Bluetongue (BT) has been diagnosed in the Netherlands. The clinical symptoms of BT on five farms during the first outbreak ever in the Netherlands are described. Fever and swollen sensitive coronets leading to reluctance to stand and walk were sometimes the first symptoms. Later lesions in the mouth occurred with foamy salivation and respiratory problems. In other cases a swollen head with swollen lips and foamy salivation were the first clinical signs. Also sudden death occurred. In the first sixteen confirmed cases morbidity and mortality were lower than described in outbreaks in other countries. Good collaboration between practitioners, specialists of the Animal Health Service (GD-Deventer), and specialists of the Food and Consumer Product Safety Authority (VWA) and CIDC-Lelystad (Wageningen UR) led to a rapid notification and ultimately confirmation of the suspected diagnosis BT.     

α‐6 Integrin Expression in Bovine Spermatogonial Cells Purified by Discontinuous Percoll Density Gradient

The study of spermatogonial stem cells (SSCs) provides a model to better understand adult stem cell biology. Besides the biomedical potential to perform studies of infertility in many species, SSCs hold a promising application at animal transgenesis. Because stem cells are thought to be associated with basement membranes, expression of α‐6 integrin has been investigated as a marker of type A spermatogonial cells, which are considered SSCs because of their undifferentiated status and self‐renewal ability. In this manner, the aim of this study was to isolate type A SSCs from adult bulls by a two‐step enzymatic procedure followed by a discontinuous Percoll density gradient purification and verify the expression of α‐6 integrin by flow cytometry and real‐time RT‐PCR before and after Percoll purification. Spermatogonial cells were successfully obtained using the two‐step enzymatic digestion. An average of 1 × 10⁵ viable cells per gram of testis was isolated. However, the discontinuous Percoll did not purify isolated cells regarding α‐6 integrin expression. Flow cytometry analysis demonstrated no differences in the α‐6 integrin expression between cell samples before and after Percoll purification (p = 0.5636). The same was observed in the real‐time PCR analysis (p > 0.05). In addition to α‐6 integrin, the expression of GFRa‐1 and PGP9.5, known bovine SSCs markers, was detected in all samples studied. Considering that Percoll can reduce cell viability, it is possible to conclude that Percoll density gradient is not suitable to purify bovine SSC, according to α‐6 integrin expression.     

Muscle reorganisation through local injection of stem cells in the diaphragm of mdx mice

Background

The diaphragm is the major respiratory muscle affected by Duchenne muscular dystrophy (DMD) and is responsible for causing 80% of deaths. The use of mechanical forces that act on the body or intermittent pressure on the airways improves the quality of life of patients but does not prevent the progression of respiratory failure. Thus, diseases that require tissue repair, such as DMD, represent a group of pathologies that have great potential for cell therapy. The application of stem cells directly into the diaphragm instead of systemic application can reduce cell migration to other affected areas and increase the chances of muscle reorganisation. The mdx mouse is a suitable animal model for this research because its diaphragmatic phenotype is similar to human DMD. Therefore, the aim of this study was to assess the potential cell implantation in the diaphragm muscle after the xenotransplantation of stem cells.

Methods

A total of 9 mice, including 3 control BALB/Cmice, 3 5-month-old mdx mice without stem cell injections and 3 mdx mice injected with stem cells, were used. The animals injected with stem cells underwent laparoscopy so that stem cells from GFP-labelled rabbit olfactory epithelium could be locally injected into the diaphragm muscle. After 8 days, all animals were euthanised, and the diaphragm muscle was dissected and subjected to histological and immunohistochemical analyses.

Results

Both the fresh diaphragm tissue and immunohistochemical analyses showed immunopositive GFP labelling of some of the cells and immunonegativity of myoblast bundles. In the histological analysis, we observed a reduction in the inflammatory infiltrate as well as the presence of a few peripheral nuclei and myoblast bundles.

Conclusion

We were able to implant stem cells into the diaphragm via local injection, which promoted moderate muscle reorganisation. The presence of myoblast bundles cannot be attributed to stem cell incorporation because there was no immunopositive labelling in this structure. It is believed that the formation of the bundles may have been stimulated by cellular signalling mechanisms that have not yet been elucidated.