Immunohistochemical Detection of Mycoplasma agalactiae in Formalin‐Fixed,Paraffin‐Embedded Tissues from Naturally and Experimentally Infected Goats

Samples from the mammary tissue of 14 lactating goats (12 naturally infected and two experimentally infected) were examined for the presence of Mycoplasma agalactiae. A monoclonal antibody (5G12) was applied to formalin‐fixed, paraffin‐wax‐embedded sections and labelled by the avidin–biotin peroxidase complex (ABC) method. Histological examination of tissue sections revealed strong immunoreactivity in all animals included in the study. Mycoplasma agalactiae antigen was mainly detected in the cellular debris at the periphery of purulent exudates present within lactiferous sinuses, and lactiferous and interlobular ducts. In addition, M. agalactiae organisms appeared in the cytoplasm of the epithelium of ducts, and in infiltrating macrophages and neutrophils within the ducts, alveoli, interstitial tissue and regional lymph node sinuses. It is concluded that this monoclonal antibody‐based immunohistochemical technique is an efficient and specific method for the post‐mortem detection of M. agalactiae in cases of clinical mastitis as well as being a useful tool for the study of the route of infection and cellular types involved during mastitis caused by this organism.     

Demonstration of Listeria monocytogenes by immunohistochemistry in formalin-fixed brain tissues from natural cases of ovine and bovine encephalitis

In the present work, evidence of Listeria monocytogenes antigens based on the avidin-biotin complex (ABC) immunoperoxidase technique was performed on formalin-fixed central nervous system tissues (CNS) from a total of 23 natural cases of encephalitis (four ovine and 19 bovine). Listeria monocytogenes serotype 4 was isolated from 10 of 17 cultured specimens. Meningoencephalitis characterized by focal necrosis, microabscesses, perivascular cuffing, and gliosis with presence of macrophages and/or neutrophils was observed at histological examination. Positive L. monocytogenes antigens were successfully identified by immunohistochemistry (IHC) in the CNS of all 23 cases. Paraffin-embedded tissues assayed were stored up for 17 years. Morbidity of the outbreaks was between 0.3-3% and 0.1-1% for ovine and bovine cases, respectively. In all the ovine cases, flocks involved were under extensive grazing conditions. In nine of the 19 bovine cases (47.3%), supplementation with corn silage was used. The ABC test can help as a practical tool for the diagnosis of natural cases of L. monocytogenes encephalitis on formalin-fixed specimens from ovine and bovine.     

高效液相色谱法测定动物可食性组织中莫昔克丁残留量

建立了适用于检测畜肉及内脏中莫昔克丁残留的高效液相色谱法。样品经乙腈提取,加水混合后,C₁₈固相萃取柱净化,氮气吹干。将N-甲基咪唑-乙腈(1+1,v/v)和三氟乙酸酐-乙腈(1+2,v/v)作为衍生化试剂,65 ℃避光反应15 min进行衍生化。以乙腈+水(90+10,v /v)作为流动相,等度洗脱,荧光法测定。结果显示：莫昔克丁在线性范围内呈良好线性关系,相关系数(R^₂)大于0.999,检测限和定量限分别为2 μg/kg和5 μg/kg。该方法在以上各组织中的回收率范围为60.5 % ～ 110 %,其批间、批内变异系数范围为0.62 % ～ 15.8 %。该方法准确性强、灵敏度高,可满足实验室实际工作需求。     

The Occurrence of Pathogenic Listeria monocytogenes and Antibodies against Listeriolysin‐O in Buffaloes

The occurrence of Listeria monocytogenes in meat and milk samples, and antilisteriolysin O (ALLO) antibodies in sera of buffaloes were studied. Isolation of the pathogen was attempted from the samples by selective enrichment in University of Vermont Medium and plating onto Dominguez–Rodriguez isolation agar. The pathogenicity of the isolates was tested by Christie, Atkins, Munch Petersen test and mouse inoculation test. Of 167 meat samples 2.4 and 10.17% were positive for L. monocytogenes and Listeria sp., respectively. Of the 64 milk samples 6.25 and 26.13% were positive for L. monocytogenes and Listeria sp., respectively. A total of 284 serum samples were tested by listeriolysin O (LLO)‐based indirect enzyme‐linked immunosorbent assay of which 25.35% were found to be seropositive. The culture positivity for L. monocytogenes and detection of ALLO did not show any agreement (κ=0.035). The prevalence of pathogenic L.monocytogenes in milk and meat and the occurrence of anti‐LLO antibodies is of concern from the public health point of view.     

Confirmed Exposure to Tick‐Borne Encephalitis Virus and Probable Human Cases of Tick‐Borne Encephalitis in Central/Northern Anatolia,Turkey

Tick‐borne encephalitis virus (TBEV) is the aetiological agent of tick‐borne encephalitis (TBE), a potentially fatal central nervous system infection of humans. TBE is endemic in many areas of Europe and Asia; however, very scarce data on TBEV activity are available from Turkey. We aimed to identify TBEV exposure in healthy blood donors and the impact of TBEV in central nervous system infections in Central/Northern Anatolia. Two‐thousand four hundred and fifty four sera, collected from blood donors at Ankara, Konya, Eski?ehir and Zonguldak branches of the Turkish Red Crescent Middle Anatolia Regional Blood Center, were analysed for TBEV serosurveillance. Paired serum and cerebrospinal fluid samples from 108 patients with the diagnosis of aseptic meningitis/encephalitis of unknown aetiology were also evaluated to identify TBE and neuroborreliosis cases. Commercial enzyme‐linked immunosorbent assays and indirect immunofluorescence tests were employed for antibody detection. Forty‐seven donor samples (1.9%) were reactive for TBEV IgG. In 25 persons with IgG reactivity (53.1%), risk factors for tick‐borne infections were revealed. One sample from Zonguldak province (1/198; 0.5%) in the Black Sea region of Turkey was confirmed to possess neutralizing antibodies via plaque reduction neutralization test. TBEV IgM was detected in 9.2% (8/108) of the patients. IgM was accompanied by IgG reactivity in two persons where, in one, recent history of a tick bite was also identified. Intrathecal antibody production for TBEV could not be demonstrated. No evidence for Borrelia infections could be found. Confirmed exposure to TBEV and/or an antigenically similar tick‐borne flavivirus is documented for the first time in blood donors in Zonguldak in Northern Anatolia. Probable cases of TBE have also been identified from Central Anatolia. The epidemiology of TBEV activity in Turkey needs to be assessed and benefits of vaccination for general population, risk groups or travellers must be considered.     

高效液相色谱-荧光法测定牛羊组织中阿苯达唑及其代谢物的残留量

建立了同时测定牛羊组织中阿苯达唑及其代谢物的高效液相色谱-荧光检测方法。试样中待测物经乙酸乙酯提取,酸性氧化铝固相萃取柱和MCX固相萃取柱净化,高效液相色谱-荧光法测定,以外标法定量。药物在5~2000μg/L浓度范围内线性关系良好,相关系数均大于0.999。方法的检出限为0.025 mg/kg,定量限为0.05 mg/kg,在不同组织中添加浓度范围为0.05~10 mg/kg,其平均回收率为60.6%~119.1%,相对标准偏差在1.7%~19.1%之间。方法抗干扰能力强、灵敏度高,适用于牛羊组织中阿苯达唑及其代谢物残留量的测定。     

Isolation of Listeria monocytogenes from buffaloes with reproductive disorders and its confirmation by polymerase chain reaction

Listeria monocytogenes, a gram-positive, facultative intracellular pathogen was isolated from buffaloes with a history of reproductive disorders and polymerase chain reaction (PCR) analyses for the presence of virulence-associated genes were conducted. A total of 530 samples of faecal, nasal, vaginal swabs and blood samples from 135 buffaloes were screened. The prevalence of L. monocytogenes and other Listeria spp. was found to be 4.4 and 7.4%, respectively. All isolates were subjected to PCR for virulence-associated genes (prfA, plcA, hlyA, actA and iap) and to pathogenicity testing by the phosphatidylinositol phospholipase C (PI-PLC) assay and mice and chick-embryo inoculation. All L. monocytogenes isolates were hemolytic and positive for the hlyA gene. One L. monocytogenes isolate possessed all five virulence-associated genes and was also positive in the PI-PLC assay as well as in the in vivo pathogenicity tests. The remaining hemolytic L. monocytogenes isolates lacking the plcA gene and PI-PLC assay activity were, however, non-pathogenic via mice and chick-embryo inoculation tests, in spite of having the hlyA gene. The detection of multiple virulence-associated genes, in combination with in vitro pathogenicity tests, must be performed to identify pathogenic L. monocytogenes.     

哈尔滨市鲜肉中单核细胞增生性李斯特菌的分离鉴定及耐药性分析

为了解哈尔滨市单核细胞增生性李斯特茵(Lm)的污染状况及耐药状况.在哈尔滨市市场随机采集158份鲜肉样品,采用显色培养基分离,API试剂条和PCR鉴定等方法对样品中的Lm进行分离鉴定,并通过Kirby-Barer法测定分离菌株对24种抗生素的耐药性.结果从鲜肉中共分离到Lm 23株,检出率为14.56%,其中鲜猪肉检出率最高,达20.00%(14/70);23株分离菌株中耐药菌株为22株,耐药率高达95.65%.这表明哈尔滨市鲜肉中存在一定程度的Lm污染,并且分离菌株存在较严重的耐药现象.应加强控制动物饲料亚治疗抗生素的使用并严格遵守休药期,防止耐药菌株产生进而控制食源性疾病的发生.     

Antibody‐Dependent Killing of Virus‐Infected Targets by NK‐Like Cells in Bovine Blood

Natural killer (NK) cell lysis of target cells by an Fc receptor‐mediated mechanism has not been conclusively demonstrated in cattle (Campos and Rossi, Vet. Immunol. Immunopathol. 8, 351–362, 1985), although it is well recognized in other species (Sulica et al., Nat. Immun. 14, 123–133, 1995). To resolve this problem, bovine peripheral blood mononuclear cells were completely depleted of adherent monocyte/macrophage type cells. The resulting enriched population of lymphocytes, was totally devoid of adherent monocytes, but contained up to 2 % NK‐like cells. On their own, this population had very low background levels of cytotoxicity for virus‐infected target cells in ⁵¹chromium release assays, but following the addition of virus‐specific antibodies, high levels of lysis were observed. This enhanced level of antibody‐dependent cytotoxicity demonstrated that bovine NK‐like cells can mediate killing of targets by an Fc receptor‐mediated mechanism as has been demonstrated for NK cells from other species.     

Avirulence of viable but non-culturable Listeria monocytogenes cells demonstrated by in vitro and in vivo models

The virulence of Viable But Non-Culturable (VBNC) cells of 4 strains of Listeria monocytogenes was investigated in both a human adenocarcinoma cell line (HT-29) and a mouse model. LO 28, ATCC 19115 and CNL 895807 strains of Listeria monocytogenes became VBNC when incubated in microcosm water at 20 degrees C and Scott A strain at 4 degrees C. No culturable bacteria were detected in the VBNC state, although 104 active cells/mL were found by the Direct Viable Count (DVC) and CTC-DAPI double staining methods. A comparison of virulence in both human adenocarcinoma cell line HT-29 and the mouse model showed that culturable controls were more virulent than VBNC cells, which appeared to be avirulent regardless of the virulence methods applied. Pathogenicity was tested in each model and was lost concomitantly with culturability, whereas some cells were still metabolically active (determined by CTC and DVC). Moreover, amplification of a 388 bp fragment with Immunocapture-PCR revealed the presence of Listeria monocytogenes DNA in all mixed spleen samples after intravenous injection of VBNC cells. These results demonstrate that VBNC cells were present in the mouse spleens. The results of the study suggest that Listeria monocytogenes strains might remain in the aquatic environment for prolonged periods in the VBNC state but these cells were not pathogenic in the conditions tested. These findings demonstrate the value of VBNC studies and show the need to investigate the role of VBNC cells in environmental transmission of Listeria monocytogenes. Further studies are needed in order to investigate the virulence of VBNC cells of Listeria monocytogenes after recovery of a culturable state.     

Clinical and histopathological aspects of naturally occurring mastitis caused by Listeria monocytogenes in cattle and ewes

Listeria monocytogenes was isolated from the milk of two cows and two sheep with mastitis in one quarter and one udder half. The animals were observed over a period of 2-12 months. Clinical examination of the udder, bacteriological examinations and determination of somatic cell counts of milk samples were performed monthly. All four cases suffered from a subclinical mastitis characterized by an elevated somatic cell count (0.8-10.1 x 10(6) cells/ml), a persistent shedding of Listeria and by a normal appearance of the milk. The animals did not show any systemic reaction, but all animals developed an atrophy of the infected mammary gland. Histological examinations revealed a chronic interstitial mastitis with diffuse infiltration of lymphocytes, plasma cells and macrophages. All internal organs showed no abnormalities, no Listeria could be isolated. Listeria could however be isolated from the affected mammary parenchyma and from the mammary lymph node. The results of the bacteriological examination could be confirmed by means of PCR. Using PFGE, all the isolates from the same animal were identical. Immunohistochemical examination of the ovine mammary glands achieved a very strong immunoreactivity for CD5 cells. The mode of infection and the reaction of the immune system's defense of the ovine udders are discussed.     

Validation of Bovine Oestrous‐Specific Synthetic Molecules with Trained Scent Dogs; Similarities Between Natural and Synthetic Oestrous Smell

Oestrous detection is crucial for successful dairy cow reproduction. Bulls identify cows in oestrus by oestrous‐specific odours especially in urine and vaginal fluid. These have been used to train dogs to detect cows in heat. To improve and simplify the dog training, a spray containing synthetic oestrous molecules was developed. The objective of this study was to test the spray on similarities to the natural substance thus to assess its suitability as a training substance for heat detection dogs. Ten privately owned dogs of various breeds were trained. Dogs should be trained either to differentiate natural vaginal fluid from cows in oestrus and dioestrus (n = 5), or spray with or without synthetic oestrous molecules (n = 5). Dogs trained on natural fluid and on spray could detect the oestrous odour they had been trained on with an overall accuracy of 69.0% and 82.4%, respectively (p = 0.019). To validate the synthetic molecules, dogs trained with synthetic molecules had to detect oestrous odour in natural fluid without further training (accuracy 37.6%). Dogs trained on natural fluid detected the synthetic molecules with an accuracy of 50.0% (50% vs 37.4%, p < 0.05). Dogs can recognize natural vaginal fluid from cows in oestrus after they have been trained with synthetic oestrous molecules, but accuracy needs to be improved.     

2005-2014年新疆生产建设兵团第三师牛羊布鲁氏菌病血清学检测与分析

布鲁氏菌病是由布鲁氏菌属细菌引起的一种人畜共患传染病。新疆生产建设兵团第三师图木舒克市地处布鲁氏菌病高发区,2005-2014年每年均开展了布鲁氏菌病检测工作。本文对该区近10年牛、羊布鲁氏菌病的血清学监测结果进行汇总,分析牛、羊布鲁氏菌病的感染动态,指出近几年布鲁氏菌病防控工作中存在的问题,为下一步采取有效措施提供参考。     

Expression of Cyclins and Cyclin‐Dependent Kinase Inhibitors in Granulosa Cells from Bovine Ovary

The regulation of granulosa cell proliferation is complex, and it is essential for normal follicular development in mammals. The aim of this study was to examine the expression of cyclins and their inhibitors in the granulosa cells of follicles at different developmental stages. Follicles were classified into three groups: oestrogen‐inactive dominant follicles (EIDs), oestrogen‐active dominant follicles (EADs) and pre‐ovulatory follicles (POs). The expression of CCND2 (cyclin D2) mRNA was significantly higher in granulosa cells from EADs and POs than in those from EIDs. The expression of CCND3 (cyclin D3) mRNA was significantly higher in granulosa cells from EADs than in those from other follicles. CCND1 (cyclin D1), CCNE1 (cyclin E1) and CCNE2 (cyclin E2) mRNA expression did not differ among the different follicular stages. The expression of CDKN1A (p21^cip1) and CDKN1B (p27^kip1) mRNA was significantly higher in granulosa cells from EIDs and POs, respectively, than in those from other follicles. Expression of CDKN2D (p19^INK4d) mRNA did not differ among the different follicular stages. Taken together, our study suggested that cyclins and their inhibitors are associated with granulosa cell proliferation at specific follicular developmental stages.     

Anti‐microbial Susceptibility of Streptococcus spp. Isolated from Bovine Mastitis in Argentina

The in vitro susceptibility to penicillin G, erythromycin and clindamycin was determined by the disc diffusion test and by E‐test for a total of 47 streptococcal strains (three Streptococcus uberis, 36 Streptococcus agalactiae, eight Streptococcus dysgalactiae spp. dysgalactiae) isolated from bovine intramammary infections in Argentina. Moreover, resistance phenotypes of erythromycin‐resistant streptococcal isolates was characterized. MIC₉₀ of penicillin G, erythromycin and clindamycin for S. agalactiae were 0.75, 8.0 and 12.0 μg/ml respectively. Resistance to erythromycin and clindamycin was detected in 13 (27.6%) and 12 (25.5%) isolates respectively. No isolate was resistant to penicillin G. Resistance against macrolides, lincosamides and streptogramin B (MLS_B) represented by the constitutive MLS_B phenotype was present in 11 (23.4%) erythromycin‐resistant isolates and two isolates (4.3%) expressed the M phenotype. The inducible MLS_B phenotype was not identified. Results suggest that beta‐lactams are the first‐line antibiotics when treating streptococcal udder infections; however, the continuous monitoring of the antibiotic resistance is essential, as the emergence of resistant strains has become a growing concern on the therapy of bovine mastitis.     

Listeriosis in sheep. Isolation of Listeria monocytogenes from organs of slaughtered animals and dead animals submitted for post-mortem examination

The udders from 13 culled ewes and liver, spleen, kidney, lung and brain from 15 lambs, 11 months old, were examined for the presence of Listeria monocytogenes (Lm) at slaughter. Lm was isolated from 1 of 13 udders, from 6 of the 15 brains and from 0–4 of the other organs from each of the 15 lambs.Internal organs from 68 sheep submitted for post-mortem examination were examined in the same way. Lm was isolated from 25 of these animals. Lm was isolated from the brain of 7 of 9 animals with encephalitis, and from 0–3 of the other 4 organs examined. Lm was also isolated from 10–20 % of the organs from animals with other diagnoses. Altogether 9 of 10 animals with encephalitis and 16 of 58 with other diagnoses (28 %) were found to harbour this organism.     

Septicaemia, encephalitis and abortions in a housed flock of sheep caused by Listeria monocytogenes type 1/2

One hundred and ninety-six housed pregnant ewes were fed poor quality silage for two days. Ewes are reluctant to eat the silage and within 48 hours they became dull and developed diarrhoea and lameness. Despite treatment with antibiotics and calcium borogluconate 19 ewes died, more than 60 developed vaginal discharges and at lambing 94 ewes were barren. Six developed nervous signs and two of these died, one with lesions typical of listeric encephalitis. Post mortem lesions and the isolation of Listeria monocytogenes type 1/2 from lung, liver, spleen and kidney are described. L monocytogenes was also isolated from blood samples from live ewes. The estimated gross financial loss to the farmer was 5130 pounds or, for the flock, 26 pounds per ewe.