Use of PCR-RFLP to identify Leishmania species in naturally-infected dogs

Tissue imprints on Giemsa stained slides from dogs were used to investigate the presence of Leishmania amastigotes by either optical microscopy (OM) or Polymerase chain reaction (PCR) detection of DNA. Samples from skin, spleen, lymph node, liver and bone marrow from a Leishmaniasis endemic area dogs where Leishmania (Leishmania) chagasi and Leishmania (Viannia) braziliensis are sympatric were studied. Dogs were initially diagnosed by Indirect Immunofluorescence (IIF), as which 39 were IIF positive (≥1:40) and 16 negative. The IIF positive dogs were clinically grouped as symptomatic (n = 15), oligosymptomatic (n = 12) and asymptomatic (n = 12). Although PCR positivity was higher in symptomatic dogs, specially their skin samples, there was no significant difference among clinical groups or organs examined. Ten (62.5%) out of 16 IIF and OM negative animals were positive for PCR in at least one organ. Forty-eight positive PCR amplicons were further submitted to RFLP for Leishmania identification. All dogs were infected with L. (L.) chagasi except one, infected with L. (V.) braziliensis. PCR was more efficient than IIF and OM to diagnose canine visceral Leishmaniasis (CVL), regardless of the organ examined and the clinical form present. The use of PCR together with serology helps determining the extension of sub clinical infection in CVL endemic areas and provides a better estimate of the number of dogs to be targeted for control measures. In conclusion, our data reinforce the need for a specific diagnosis of canine infection in areas where diverse Leishmania species are sympatric and demonstrate that PCR–RFLP can be used to identify Leishmania species in dog tissue imprint stained slides.     

Zinc supplementation increases resistance to experimental infection by Trypanosoma cruzi

It is well recognized that zinc is an essential trace element for all organisms, influencing growth and affecting the development and integrity of the immune system. It is also well known that the protective response against Trypanosoma cruzi depends on both innate and acquired immunity and for the control of the parasite load and host survival, the participation of special cells such natural killer (NK), T and B lymphocytes and macrophages are required. So the aims of this study were to evaluate the effects of zinc supplementation on the host's immune response infected with T. cruzi. Our data point in the direction that zinc supplementation triggered enhanced thymocyte and splenocyte proliferation as compared to unsupplied group of animals. It is also important to emphasize that interleukin-12 (IL-12) participates in the resistance to several intracellular pathogens including T. cruzi. Our findings demonstrate an enhanced production of IL-12 during the acute phase of infection in zinc-supplied groups. So we conclude that zinc supplementation leads to an effective host's immune response by up-modulating the host's immune response, thus contributing in the reduction of blood parasites and the harmful pathogenic effects of the experimental Chagas’ disease.     

Prevalence of Surra among camels and horses in Jordan

The prevalence of Trypanosoma evansi infection among camels and horses in Jordan was studied using thick blood smears and inoculation techniques with mice and rats. A total of 437 camels and 83 horses from four climatic zones were surveyed. In addition, 40 donkeys, 32 cattle and 35 goats in contact with infected camels and horses were also tested in the same way. Clinical disease was evident in 8.2% of the camels (36 out of 437) and in 9.6% of the horses (8 out of 83). Infection was limited only to the Sweama area on the Dead Sea (within the warm desert-climatic zone), with prevalence of 30.5% and 33.3%, respectively, for camels and horses. Donkeys, cattle and goats examined were all free from T. evansi. Clinically affected camels were positive by both, thick blood smear and mouse and rat inoculations. Rat and mouse inoculations revealed (X²=3.2, df=1, exact p=0.07) greater number of positive cases in horses than those revealed by thick blood smears. T. evansi-infected camels and horses showed all the clinical signs known for Surra. In addition, it was observed that 100% of infected camels stared at the sun.     

The Isolation,Identification and Phylogenetic Analysis of Theileria

Bovine theileriosis was a kind of hemo-protozoan diseases that seriously hindered the sustainable development of cattle industry.In the present study, the infection of Theileria was detected using microscopic examination and species-specifc PCR of T.annulata, T.sergenti and T.sinensis from 18 blood samples collected from a breeding cattle farm in China.Then ITS and MPSP genes were amplified and cloned using universal primers of ITS and allele-specific primers of 4 major piroplasm surface protein (MPSP) from the positive samples.The sequences were used to make alignment, polymorphism and phylogeny analysis.The results showed that 3 samples were positive for Theileria and the 3 positive samples were all the T.sergenti infection.The amplification of allelic MPSP gene of T.sergenti from the 3 positive samples showed that two of them were single infection by Ikeda-type and another one was co-infection with both Chitose-type and Ikeda-type, while Buffeli-type and Thai-type were not detected.Moreover, on the basis of phylogenetic tree constructed with MPSP gene sequences, types 1 and 2 of MPSP were confirmed to be present in the cattle farm.The results revealed that there were T.sergenti infection in the breeding cattle farm, and these parasites at least with 2 allelic MPSP gene types were present, which indicated that the immune prevention and control of the disease became more complicated.Our research laid foundation of the further study on T.sergenti infection and disease prevention and control.     

牛泰勒虫的分离鉴定及进化分析

牛泰勒虫病是严重危害养牛业可持续发展的血液原虫病,本试验采用血涂片镜检和特异性PCR检测技术,对中国某种牛场的18份血样进行了泰勒虫检测,然后分别用ITS基因通用引物和4种MPSP等位基因特异性引物自阳性样品中扩增出对应的基因,克隆测序后,进行序列比对和进化分析,确定泰勒虫基因型。结果显示,自18份牛血样中检出3份阳性样品,且全为瑟氏泰勒虫感染;3个阳性样品的瑟氏泰勒虫MPSP等位基因扩增结果显示,1个阳性样品为I (Ikeda) 和C (Chitose)型的混合感染,另2个样品为I型单一感染,而均无B (Buffeli)和Thai型;用扩增的MPSP基因测序,构建进化树,确认其感染的瑟氏泰勒虫存在MPSP 1型和2型。这些结果表明,该种牛场存在瑟氏泰勒虫感染,且同时存在2种MPSP等位基因型;MPSP等位基因的复杂性可能使该病的免疫防控更加困难。本试验结果为深入研究瑟氏泰勒虫感染情况及免疫防控提供了数据支持,并为养防一体做好监控。     

Plasma and tissue concentrations and molecular forms of somatostatin in calves infected with Sarcocystis cruzi

The effects of parasitic infection on plasma and tissue content of immunoreactive somatostatin (SRIF) were studied in 4-mo old male calves inoculated with the protozoan Sarcocystis cruzi. Because feed intake significantly decreased (70%) in infected calves around day 28 postinfection (pi), concomitant with the asexual replication of S. cruzi and outward expression of clinical signs, the relative contributions of infection and associated reduction in nutrition on plasma SRIF were evaluated. Treatment groups were: noninfected ad libitum fed (C), infected (250,000 S. cruzi oocysts per os) ad libitum fed (I) and noninfected calves pairfed to the level of intake of each infected calf (PF). Mean plasma concentrations of SRIF (pg/ml) on day 30 pi were: C, 224 ± 22; I, 742 ± 150; PF, 246 ± 31 (effect of infection P<.05). In another study, SRIF was measured in plasma and in pancreatic, duodenal, jejunal and ileal tissue extracts from normal and S. cruzi infected calves. Plasma and tissue samples were collected on day 42 pi. Mean plasma SRIF were 2.5 times higher in infected than control calves. Plasma insulin and insulin-like growth factor 1 was lower in infected v control calves (P<.02). Plasma glucagon was similar between groups. Duodenal (P<.05) and jejunal (P<.02) SRIF content was higher in infected than control calves. Chromatography of tissue extracts on Sephadex G-50 revealed that the increase in SRIF was accounted for, in part, by molecular forms larger than cyclic SRIF-14. Data suggest that peripheral SRIF is increased in calves during S. cruzi infection. The increase in SRIF is not solely related to plane of nutrition. Altered levels of gut SRIF(s) may be associated with perturbed metabolic regulation in parasitized animals through direct effects on the gut.     

Blastomycosis in cats: five cases (1979-1986)

Medical records of 5 cats with blastomycosis examined at the University of Tennessee Veterinary Teaching Hospital from 1979 to 1986 were reviewed. Clinical signs of blastomycosis varies depending on the organ(s) affected, but respiratory tract disease was most common, followed by CNS signs and ocular problems. A definitive diagnosis was made by identification of characteristic fungal organisms in biopsy or necropsy specimens. Two cats treated with amphotericin B did not respond to treatment and died or were euthanatized. The lungs, brain, eyes, and lymph nodes commonly were affected, but one cat had only cutaneous and regional lymph node involvement. The respiratory tract appeared to be a common primary site of infection, with dissemination to other organ systems. The typical host response was a pyogranulomatous cellular infiltrate with numerous fungal organisms evident.     

Trypanosoma evansi infection in worked and unworked buffaloes (Bubalus bubalis) in Indonesia

The effect of controlled amounts of exercise on the outcome of Trypanosoma evansi infection was studied in groups of swamp buffaloes (Bubalus bubalis) experimentally infected with T. evansi. Daily body temperature, packed cell volume (PCV) and parasitaemia measurements were obtained from each animal for up to 110 days after infection. Exercise did not appear to exacerbate the effect of T. evansi infection in that similar temperature, PCV and parasitaemia profiles were obtained with both exercised and rested animals. Trypanosoma evansi infection, however, had a marked effect on temperature and PCV profiles, both of which could adversely affect an infected animal's work output and work tolerance.     

Investigation of Molecular Etiology of Theileria equi Infection in Three Herds

To explore the prevalence of Theileria equi (T.equi) infection in horse in Guizhou province, the antibody level and 18S rRNA gene were detected from blood samples of Guizhou pony, Southwest horse and Yili horse using competitive ELISA and PCR methods.Giemsa-stained blood smear was prepared to observe T.equi in red blood cells.Intact protozoans of T.equi were observed in red blood cells of horses at Giemsa-stained slide smears with a detection rate of 12.5%.The 18S rRNA gene fragment of T.equi was detected in Guizhou pony, Southwest horse and Yili horse, and the consistent rates with the known nucleotide sequence were 97% to 100%.The PCR result indicated that the positive rates of T.equi in Guizhou pony (76.62%) and Yili horse (73.81%) were similar, which were higher than that in Southwest horse (33.33%).Furthermore, the antibody levels against T.equi in Guizhou pony (24.68%) and Southwest horse (12.12%) were lower than that in Yili horse (31.71%).A weak correlation between the antibody level and the blood physicochemical indexes was calculated from Guizhou pony and Southwest horse, including weak positive correlations with neutrophils numbers, gamma-glutamyl transferase and creatine kinase levels, and weak negative correlations with the numbers of red blood cell, white blood cell, platelet and lymphocyte and contents of hemoglobin.It suggested that a higher proportion of T.equi infection present in three herds.     

3个马群感染马泰勒虫的分子病原学调查

试验采用显微镜观察、PCR和竞争性酶联免疫吸附试验(competitive enzyme-linked immunosorbentassay,cELISA)等方法对贵州矮马、西南马和伊犁马的马泰勒虫病的感染状况进行研究。结果显示,从32份新鲜的血液涂片中,观察到形态完整的马泰勒虫(Theileria equi)虫体,检出率12.5%。从贵州矮马、西南马及伊犁马3个马群血液总DNA中都检测到马泰勒虫的18S rRNA基因片段,与已知序列的同源性为97%~100%;相比之下,贵州矮马与伊犁马的阳性率相近,分别为76.62%和73.81%,西南马较低,仅为33.33%。另外,经cELISA检测,与伊犁马(31.71%)相比,贵州矮马和西南马血液中抗马泰勒虫抗体的阳性率较低,分别为24.68%和12.12%,并与两个马群的血液理化指标存在一定的联系:与中性细胞数量、γ-谷氨酰转移酶和肌酸激酶的含量呈弱正相关;与红细胞、白细胞、血小板、淋巴细胞数量及血红蛋白含量呈弱负相关。这些研究结果提示3个马群中均存在较高比例的马泰勒虫感染。     

弓形虫和新孢子虫交叉反应抗原MIC7A对小鼠的免疫保护效力分析

弓形虫和新孢子虫是两种亲缘关系接近的顶复亚门原虫,二者之间存在一定程度的交叉免疫保护作用,这种作用可能是基于交叉反应抗原产生的。本研究旨在表达并鉴定弓形虫和新孢子虫的交叉反应抗原TgMIC17A,通过将其应用于小鼠的免疫保护试验,评估该抗原对弓形虫和新孢子虫感染产生的交叉免疫保护作用。对TgMIC17A进行基因克隆和原核表达,通过免疫印迹试验鉴定其反应原性和交叉反应性。重组蛋白免疫小鼠后测定血清特异性IgG抗体水平,评价其免疫原性。二免后,分别用1×10³个弓形虫Pru速殖子、1.5×10⁷个新孢子虫Nc1速殖子攻虫,对小鼠的体重变化、存活率进行监测,并在攻虫30 d后检测各组存活小鼠的脑荷虫量,评价TgMIC17A重组蛋白免疫小鼠后对弓形虫和新孢子虫的交叉免疫保护效果。结果显示,rTgMIC17A可以被弓形虫和新孢子虫的阳性血清识别,相较于未免疫组小鼠,免疫组小鼠体内可产生高水平特异性IgG抗体(P＜0.01),且感染弓形虫或新孢子虫的脑荷虫量均显著降低(P＜0.01)。本研究克隆并表达了TgMIC17A,鉴定其为新孢子虫和弓形虫的交叉反应抗原。该抗原可以刺激小鼠产生较好的体液免疫反应,并对弓形虫和新孢子虫的感染产生一定的交叉免疫保护作用,可以为弓形虫和新孢子虫共感染的防治,以及筛选具有交叉免疫保护力的重组疫苗提供可借鉴的研究资料。     

中国不同地区羊吕氏泰勒虫分子流行病学调查及遗传进化分析

为了解吕氏泰勒虫在中国不同地区山羊和绵羊中的流行情况,本研究应用基于吕氏泰勒虫18S rRNA基因位点的PCR检测方法,对采自中国河南、甘肃、陕西、山西、贵州、云南、新疆7个地区的281份羊血液样品进行检测,并对阳性样品测序以进行序列分析。结果显示,羊吕氏泰勒虫总感染率为35.23%（99/281）。不同采样点羊吕氏泰勒虫感染率差异极显著（P<0.01）,其中河南省羊吕氏泰勒虫感染率最高（98%,49/50）,新疆最低（0）。绵羊和山羊吕氏泰勒虫感染率分别为51.67%（31/60）和30.77%（68/221）,差异极显著（P<0.01）;放牧羊吕氏泰勒虫感染率（41.55%,86/207）极显著高于舍饲羊（17.57%,13/74）（P<0.01）;羊吕氏泰勒虫感染率在春、夏、秋及冬四季分别为56.00%（28/50）、42.75%（59/138）、9.43%（5/53）和17.50%（7/40）,差异极显著（P<0.01）;≥ 12月龄和<12月龄羊吕氏泰勒虫感染率分别为33.50%（67/200）和39.51%（32/81）,差异不显著（P>0.05）。此外,遗传进化分析表明,本研究获得的羊吕氏泰勒虫分离株与中国流行的吕氏泰勒虫分离株同源性高达99.60%以上,且位于同一分支上。本研究为进一步了解中国不同地区羊吕氏泰勒虫的流行现状及分布提供了重要参考依据。     

Fibrosarcoma with lung and lymph node metastases in an Asian elephant (Elephas maximus).

A case of fibrosarcoma with lung and lymph node metastases in a 54-year-old female Asian elephant (Elephas maximus) is described. After pododermatitis of 2 years duration in the right forefoot, a mass developed in the lateral toenail. At postmortem, metastasis to the right axillary lymph node and both lungs was noted. Microscopic examination of primary and metastatic sites revealed infiltrating bundles of spindle cells, with fairly distinct cell borders, variable amounts of eosinophilic cytoplasm, and elongate or oval nuclei. Tumor cells were often arranged in interwoven bundles and herringbone patterns. Mitotic figures were numerous and frequently bizarre. The diagnosis of fibrosarcoma with lung and lymph node metastases was made on the basis of histologic features and positive immunohistochemical staining for vimentin.     

Hypereosinophilic paraneoplastic syndrome in a cat with intestinal T cell lymphosarcoma

A 10-year-old, neutered female, domestic shorthair cat was presented with a recent history of weight loss, polydipsia, diarrhoea and vomiting. On physical examination, intestinal thickening and mesenteric lymph node enlargement were apparent. Clinical investigations revealed peripheral blood eosinophilia, eosinophilic abdominal effusion and eosinophilic mesenteric lymphadenitis. There was a temporary response to treatment with glucocorticoids but signs progressed and the cat was euthanased. On histology, there was eosinophilic infiltration and fibroplasia of intestine and mesenteric lymph nodes. Large aggregates of neoplastic round cells in the intestine and lymph nodes were identified as T lymphocytes using immunohistochemistry. A diagnosis of intestinal T cell lymphosarcoma was made. This case demonstrates that hypereosinophilic paraneoplastic syndrome may occur in cats with lymphosarcoma. Eosinophil chemotaxis may be a response to the production of interleukin-5 by neoplastic lymphocytes.     

犬弓首蛔虫感染比格犬不同阶段肝circRNAs表达模式的分析

旨在探究终末宿主肝环状RNA （circRNAs）在犬弓首蛔虫诱导致病过程中的潜在调控作用。18只6~7周龄的比格犬被平均分为3组（感染后0.5 d组、感染后1 d组和感染后36 d组），每组包括感染和对照各3只，分别于感染后对应的时间点收集肝样品。提取感染和对照组肝的总RNA，利用高通量RNA测序，构建circRNA文库，对差异表达的circRNAs进行分析，并对差异表达circRNAs的亲本基因进行GO注释和KEGG富集分析。随机选取9个差异表达circRNAs进行实时荧光定量PCR验证。结果显示，与对照组相比，在感染后0.5、1和36 d分别有94、103和84个犬肝的差异表达circRNAs。Venn图显示，3个感染阶段没有共同表达的差异circRNAs；GO和KEGG分析显示，差异表达的circRNAs参与了宿主肝免疫与炎症的相关通路，其中差异表达的circRNA novel_circ_0016108、novel_circ_0016184和novel_circ_0027468与犬弓首蛔虫引起的天然免疫相关；novel_circ_0002212参与了犬弓首蛔虫引起的肝炎症反应；novel_circ_0002212和novel_circ_0019907在宿主肝抵抗犬弓首蛔虫侵袭的过程中发挥作用。RT-qPCR验证结果显示大多数差异表达的circRNAs与高通量测序结果一致。结果表明，肝circRNAs在犬弓首蛔虫感染终末宿主的过程中发挥着重要作用，这为进一步探究犬弓首蛔虫与宿主之间的相互作用提供了新的信息，并且也为促进疾病干预措施的制定提供参考。     

Clinical and serological follow-up in dogs with visceral leishmaniosis treated with allopurinol and sodium stibogluconate

Seven dogs with parasitologically proven clinical visceral leishmaniosis (Leishmania infantum infection) were treated with a combination of allopurinol and sodium stibogluconate. The dogs received first orally 15 mg/kg of allopurinol every 12 h until the clinical signs improved, in the following 1 month period allopurinol at same dose and subcutaneously 30 mg/kg of sodium stibogluconate combination were given daily and at the end of the combined treatment, allopurinol was continued alone at the same dose till the end of 8 months. During the treatment period, dogs were supported by additional proteins, vitamins, and minerals. A long acting insecticide (collar or drop) was also used in order to prevent further parasite transmission. Follow-up was maintained by clinical, clinicopathological evaluation, and parasitological examination of lymph node, serology using the indirect immunofluorescent antibody test (IFAT). Before treatment commenced, the most important clinical signs were exfoliative dermatitis, ulcerations, peripheral lymhadenopathy, pale mucous membranes, weight loss, and ocular lesions. Clinicopathological findings included commonly anaemia, hyperproteinaemia, hyperglobulinaemia and hypoalbuminaemia. Before the treatment, amastigotes were seen in six of the seven dogs by examination of lymph node aspiration, and IFAT-titers were positive in all dogs. At the end of 8 months treatment, remission of clinical signs, restoration to normal of clinicopathological abnormalities were noticed. Lymph node aspiration was performed on three out of the seven dogs at the end of the treatment because of the very small sizes of the lymph nodes, and no amastigotes were observed. Although the mean IFAT-titer of the dogs were significantly (P < 0.001) lower compared with pretreatment, IFAT-titers of dogs were still positive. No relapses occurred during treatment period and a 6-24-month duration after the end of therapy. Based on the above results, long-term use of allopurinol combined with sodium stibogluconate together with support treatment concluded to have enough therapeutic efficacies in the treatment of dogs with visceral leishmaniosis. Observations of the cases for possible relapses were still going on and insecticide application was carefully carrying on in order preventing a possible re-infection.     

Isolation of Haemophilus agni from Six Alberta Ram Lambs with Septicemia

Six ram lambs were submitted to a veterinary diagnostic laboratory for necropsy. Clinical signs included sudden illness or death with or without observed depression, reluctance to move, scours or fever. Gross findings and histopathology revealed evidence of bacterial septicemia. Haemophilus agni was isolated from brain, spleen, lung and lymph node.     

Interaction of Tritrichomonas foetus and the bovine oviduct in an organ culture model

Tritrichomonas foetus is an extracellular parasite of the reproductive tract in cattle. The mechanism by which T. foetus causes abortion in cattle is largely unknown. There are no studies of infection in the cow oviducts, almost all published papers are related to vagina infection and few articles focusing on the uterus. The aim of the present study was to establish a working model of bovine oviduct epithelial cells and submit these cells to Tritrichomonas foetus interaction. Twenty bovine oviducts were obtained from cows at a commercial abattoir and T. foetus was injected through the isthmus into the oviduct lumen. The whole oviduct was analyzed by scanning and transmission electron microscopy. The results reported here demonstrate that: (1) fresh whole oviducts can be used as a good model to study parasite-host cell interaction; (2) cow oviduct epithelium has been shown to consist of two cell types: ciliated and nonciliated secretory cells, and T. foetus displayed great specificity for the nonciliated cells localized in the deeper oviduct folds; (3) T. foetus adheres as single separate cells, and maintains the flagella externalized; (4) differently from T. vaginalis, T. foetus does not change its shape during the adhesion process; and (5) oviduct cells exhibited morphological characteristics of apoptosis after trichomonadal interaction.     

A femorotibial joint swelling with popliteal lymph node enlargement in a Rottweiler

A 9-year-old male Rottweiler was presented to the Veterinary Medical Hospital Montenegro, Porto, Portugal with a large mass medial to the left stifle and radiographic signs of bone lysis involving the proximal tibia, fibula, and distal femur. A fine-needle aspiration was obtained from left popliteal lymph node, which was markedly enlarged. Cytologic examination revealed a highly cellular sample consisting of pleomorphic cells with marked anisocytosis and anisokaryosis and other criteria of malignancy. Some cells contained intracytoplasmic, granular, dark brown material, consistent with hemosiderin. Histologic evaluation of the surgically-excised lymph node revealed a neoplastic proliferation of histiocytic cells, with marked pleomorphism; occasional cells were erythrophagocytic. Mitotic figures were frequently observed, and many were atypical. Histologic findings were consistent with malignant histiocytosis. Necropsy examination confirmed the diagnosis of malignant histiocytosis with systemic involvement affecting most organs examined. This case of malignant histiocytosis case had an atypical clinical presentation, mimicking a musculoskeletal disorder. It underlines the importance of cytology as a simple, inexpensive, rapid and noninvasive complementary exam in routine clinical practice that can permit early diagnosis of the disease and timely selection of the most adequate therapy.