Use of nested polymerase chain reaction (PCR) for detection of retroviruses from formalin-fixed, paraffin-embedded uveal melanomas in cats

Thirty-six formalin-fixed, paraffin-embedded enucleated globes from cats with a diagnosis of diffuse anterior uveal melanoma were obtained. Sections of tumor were excised, deparaffinized, and subjected to nested polymerase chain reaction (PCR) to identify proviral DNA sequences from the feline leukemia virus (FeLV)–feline sarcoma virus (FeSV; 36 eyes), and the feline immunodeficiency virus (FIV; 18 eyes). All samples tested were negative for FIV DNA. Three samples were positive for FeLV–FeSV DNA. This is the first reported evidence of a possible link between naturally occurring feline anterior uveal melanoma and the presence of FeLV–FeSV DNA.     

Characterisation of lymphosarcomas in Australian cats using polymerase chain reaction and immunohistochemical examination

Objective To examine tumour tissue of cats with lymphosarcoma for the presence of feline leukaemia virus and feline immunodeficiency virus and analyse the immunophenotype of the tumours.
Design A retrospective study of feline lymphosarcoma cases.
Methods Formalin-fixed, paraffin-embedded tumour tissue of 14 feline lymphosarcomas was examined for the presence of feline leukaemia virus and feline immunodeficiency virus by polymerase chain reaction and immunohistochemistry. Using polyclonal and monoclonal antibodies against T and B lymphocytes, the phenotypic expression of the tumours was characterised.
Results No feline leukaemia virus antigen or proviral sequences were detected. Feline immunodeficiency virus proviral sequences were detected in two cases by polymerase chain reaction. Immunophenotyping of all 14 cases resulted in seven cases being classified as B-cell phenotype, four as T-cell phenotype, and the remaining three undetermined.
Conclusions In contrast to previous reports overseas, our results suggest that feline leukaemia virus infection appears to be an infrequent cause of lymphosarcoma in the cats that were necropsied. Feline immunodeficiency virus may have a role in lymphomagenesis. The potential role of feline immunodeficiency virus needs to be explored in more depth. Compared with most previous reports, B-cell tumours were more common than T-cell tumours in this series of cats.     

同时检测犬瘟热病毒和犬细小病毒双重PCR方法的建立

为建立可以同时检测犬瘟热病毒(CDV)和犬细小病毒(CPV)的双重PCR方法,本研究根据GenBank登录的CDV N蛋白序列和CPV NS基因保守序列,设计合成2对特异性引物。通过优化反应条件,对CDV阳性病毒株反转录后的cDNA模板和CPV的DNA模板进行双重PCR扩增,同时得到2条与试验设计相符的669 bp(CDV)和392 bp(CPV)特异性条带,建立了同时检测CDV和CPV的双重PCR方法。实验结果表明:在同一PCR反应体系中可以同时检测这2种病毒,而对犬腺病毒Ⅰ型、犬腺病毒Ⅱ型、狂犬病毒检测均为阴性;CDV和CPV的最低检出限分别为101.8TCID50和101.4TCID50。采用该方法对在黑龙江省不同地区所采集的30份犬病料样品进行检测,CDV阳性率为30%;CPV阳性率为23.33%,表明建立的PCR方法可以用于临床诊断。     

Molecular subtyping of feline immunodeficiency virus from cats in Melbourne

OBJECTIVE: To determine the subtypes of feline immunodeficiency virus (FIV) present in the domestic cat population in Melbourne. METHODS: Blood samples were collected from 42 cats that had serum antibodies against FIV. DNA was extracted and subjected to polymerase chain reaction (PCR) to amplify variable regions of the envelope (env) and group specific antigen (gag) genes of FIV. PCR products were directly sequenced or sequenced after cloning when direct sequencing yielded ambiguous results. Phylogenetic analysis was performed and comparisons made with representative sequences of different subtypes. RESULTS: The variable region of the env gene was successfully amplified by PCR from 41 of the 42 cats. All 41 were found to cluster with subtype A env sequences. The variable region of the gag gene was successfully amplified by PCR from all 42 cats. Forty-one were found to cluster with subtype A gag genes and one was found to cluster with subtype B sequences, suggesting that it may be derived from a recombinant env A/gag B virus. CONCLUSIONS: Subtype A is the predominant FIV type in Melbourne, although a subtype A/B recombinant was identified in the population of FIV positive cats. These results of env gene analysis were similar to those in a previous Australian study, suggesting that subtype A predominates in Australia. The results of the gag gene analysis show the importance of analysing multiple areas of the FIV genome when assigning FIV subtypes. Comparison with other major urban centres may provide useful information about the phylogenic diversity of FIV in Australia.     

The morphological effects of vincristine sulfate on canine bone marrow cells

This study documents the morphologic changes observed in the bone marrow aspirate biopsies from dogs 6 and 24 hours after receiving a single therapeutic dose (0.025 mg/kg) of vincristine sulfate (Oncovin: Eli Lilly & Co., Indianapolis, Ind.) intravenously. The most striking cytologic changes were observed in the erythroid cell line. Abnormalities included increased numbers of mitotic figures, abnormal nuclear configurations, and fragmented nuclei. Erythroid cells in metaphase were prominent in marrow samples collected 6 hours post-vincristine, accounting for a mean of 27% of all erythroid precursors. Fragmented nuclei and atypical nuclear configurations were seen in low numbers (mean = 7%) of erythroid cells from these animals. In contrast, marrow collected from dogs 24 hours post-vincristine exhibited low numbers (mean = 1%) of erythroid cells in metaphase, but erythroid cells with atypical nuclear configurations and fragmented nuctei accounted for a mean of 41% of the erythroid cells present. Less dramatic increases in the number of mitotic non-erythroid cells were seen 6 hours post-vincristine (mean = 5% of non-erythroid cells) and 24 hours post-vincristine (mean = 1% of non-erythroid cells). Only rare nuclear fragmentation was observed in these cell lines. Significant alterations in megakaryocytes and myeloid to erythroid (M:E) ratios were not observed in samples taken 6 hours post-vincristine; however, M:E ratios were considerably higher in three of the four samples taken from dogs 24 hours post-vincristine. Similar time-related changes were observed in four clinical cases in which bone marrow aspirates were performed after vincristine administration.     

The use of immunohistochemistry and the polymerase chain reaction for detection of feline leukemia virus and feline sarcoma virus in six cases of feline ocular sarcoma

Ocular sarcoma was diagnosed by light microscopic examination in enucleated globes ( n  = 4), orbital tissue biopsy ( n  = 1) and ocular evisceration contents ( n  = 1) from six cats. To determine if feline leukemia virus (FeLV) or a replication-defective FeLV, feline sarcoma virus (FeSV), was present in these ocular sarcomas, immunohistochemistry (IHC) and polymerase chain reaction (PCR) for FeLV were utilized. Immunohistochemical staining for FeLV glycoprotein 70 (gp70) was performed on all six formalin-fixed, paraffin-embedded tumors using an avidin–biotin complex technique. DNA was extracted from each specimen and a 166 bp region of the FeLV long-terminal repeat (LTR) was amplified by PCR. All tumors were composed primarily of spindle cells; two neoplasms had PAS-positive basement membrane enveloping areas of spindle cells. All tumors involved the uvea and five of six tumors showed transcleral extension, one of which invaded the optic nerve. Immunohistochemical staining for FeLV gp 70 was negative. PCR to amplify a portion of the FeLV LTR was negative. Based on these findings of these limited number of cases, FeLV/FeSV may not play a role in the tumorigenesis of feline ocular sarcomas. However, additional tumors representing all morphological subtypes should be investigated for the presence of viral antigen and DNA. It is important to determine the etiology and pathogenesis of these malignant ocular sarcomas. If the cell of origin and pathogenesis involve ocular and lenticular injury, and FeLV/FeSV is not present, then the clinical management of cases of feline ocular trauma, uveitis and glaucoma may prevent the development of this tumor.     

Magnetic resonance imaging of bone marrow in the pelvis and femur of young dogs

The purpose of this study was to determine the magnetic resonance (MR) imaging characteristics of bone marrow in the pelvis and femur of normal, young dogs. Six greyhounds were imaged at 4, 8, 12, and 16 months of age. Sagittal images of the femur and dorsal images of the pelvis were obtained with T1-weighted, fast spin echo (FSE) T2-weighted, and short tau (T1) inversion recovery (STIR) sequences. On T1-weighted images areas with high signal intensity, similar to fat, included the femoral heads, mid-diaphysis of the femur, femoral condyles, and the body of the ilium. T2-weighted images were characterized by uniform intermediate signal intensity (less than fat, but greater than muscle) in the femoral head, high signal intensity, similar to fat, in the mid-diaphysis of the femur and ilial body, and intermediate to high signal intensity in the femoral condyle. By 16 months high signal intensity was seen in the diaphysis and distal metaphysis on both T1- and T2-weighted images. On STIR images the femoral head had intermediate to low signal intensity, compared with muscle. The mid-diaphysis of the femur was of low signal intensity, similar to fat, and the body of the ilium had mixed signal intensity at all ages. The femoral condyle had inhomogenous, intermediate to low signal intensity at 4 months, but was of uniform low signal intensity at 8-16 months.     

8种中药成分体外抗犬细小病毒的研究

本研究旨在观察8种中药成分黄芪多糖（APS）,白术多糖（RAPS）,黄芩多糖（BSPS）,金银花多糖（FLPS）,栀子苷（CS）,苦参生物碱（SFA）,黄芪皂苷（AS）和金银花黄酮（FLF）体外对犬细小病毒（CPV）感染细胞能力的影响。分别将8种中药成分通过先加中药再接病毒、病毒和中药感作后加入和先接种病毒再加中药3种加药方式加入到培养24h的F81细胞中,于病毒接种后72h用MTT法测定F81细胞活性以评价各中药成分对F81细胞抵抗犬细小病毒感染的影响。结果,在第1种加药方式下,APS、RAPS、BSPS、FLPS抗CPV作用较强;在第2种加药方式下CS的抗病毒作用最为显著;在第3种加药方式下CS、SFA抗病毒作用最为显著。结果表明,8种中药成分均能不同程度地促进F81细胞抵抗病毒感染,部分中药成分的抗病毒作用与加药方式有关且呈一定的量效关系。     

Genomic Typing of Canine Parvovirus Circulating in the State of Rio de Janeiro,Brazil from 1995 to 2001 Using Polymerase Chain Reaction Assay

In this study, the genomic types of canine parvovirus (CPV) circulating in the State of Rio de Janeiro, Brazil, from 1995 to 2001, were investigated using the polymerase chain reaction assay (PCR). A total of 78 faecal samples from gastroenteritic puppies, confirmed as positive for canine parvovirus by haemagglutination/haemagglutination inhibition tests or virus isolation in cell culture (MDCK), were examined. The viral DNA was extracted from faecal samples using a combination of phenol– chloroform and silica–guanidine thiocyanate methods. PCR was carried out with differential pairs of primers to distinguish the old (CPV-2) and new types of virus (CPv-2a or CPV-2b). Specific amplicons were observed for all samples using the primer pair P2ab, which detects CPV-2a and CPV-2b. Seventy-six from a total of 78 samples (97%) were considered as CPV-2b because of their reaction with the primer pair P2b. Thirty samples (30/78) were from previously vaccinated puppies and in 15 of them the enteritis symptoms began from 1 to 12 days after vaccination. PCR confirmed the infection by wild virus (CPV-2b) in 5 of these 15 puppies who had received old-type vaccines. Our results show that CPV-2b was the prevalent type circulating in the State of Rio de Janeiro from 1995 to 2001.     

南京地区犬细小病毒基因型鉴定

为调查南京地区犬细小病毒(canine parvovirus,CPV)基因型分布情况,采集南京市某宠物医院2012-2019年犬细小病毒病患犬粪便43份作为检测病料,通过PCR扩增犬细小病毒VP2基因片段,并将扩增产物测序,分析CPV基因型.结果显示,43份病料皆扩增出目的片段,经测序后使用MegAlign软件与Gen...     

犬细小病毒南京分离株基因型分析

采用PCR技术对不同来源的犬细小病毒毒株进行了基因型鉴定,发现所鉴定的1982~2004年的5株犬细小病毒南京分离株和荷兰Intervet疫苗株(CPV-V154)均为CPV-2b亚型,而且相互之间的同源性均高于98.4%。     

猫细小病毒、犬细小病毒、貂细小病毒的特征比较

猫细小病毒、犬细小病毒和貂细小病毒是3种极为相似的细小病毒。最初人们主要根据患病水貂、猫、犬临床症状相似的特点,注意到它们之间可能有密切关系。时至今日,对这3种病毒的许多方面都已进行了深入的研究。作者从猫细小病毒、犬细小病毒、貂细小病毒病共同特性、生物学差异和进化机制等方面对这3种病毒的特征进行了比较和综述。     

Prevalence and risk factors for cats testing positive for feline immunodeficiency virus and feline leukaemia virus infection in cats entering an animal shelter in New Zealand

Abstract

AIMS

To estimate the prevalence of cats testing positive for antibodies to feline immunodeficiency virus (FIV) and feline leukaemia virus (FeLV) antigens in domestic cats entering a New Zealand animal shelter, based on a commercial point-of-care ELISA, to identify risk factors associated with cats testing positive, and to compare the results obtained from the ELISA with those obtained using PCR-based testing.     

多重PCR同时检测猪圆环病毒2型,猪细小病毒,猪繁殖与呼吸综合征病毒和猪瘟病毒

本试验建立一种可同时检测猪圆环病毒2型(PCV-2),猪细小病毒(PPV),猪繁殖与呼吸综合征病毒(PRRSV),猪瘟病毒(CSFV)4种病毒的多重PCR方法.对于每一种特定的病毒,用4对寡核苷酸引物均能特异扩增其目的片段.以含有病毒目的片段的质粒为模板,测定了多重PCR的检测灵敏度,PRRSV和CSFV检测最低限是48 pg,而PPV和PCV-2为0.48 pg.利用建立的多重PCR方法对具有产自有繁殖障碍母猪的仔猪或具有呼吸障碍症状的76个仔猪样本进行检测.检出了4种病毒的存在,其中26个样本(34.2%)同时感染了2种以上病毒.结果表明多重PCR方法检测猪混合感染的病毒,是一种快速、灵敏、低成本、高效率的病原学诊断工具.     

天津某区宠物医院犬细小病毒感染调查

2010年10月-2011年4月对天津某区宠物医院就诊病犬进行了调查。结果表明,在宠物医院就诊的359只病犬中,犬细小病毒感染36只,发病率为10%,治愈率为27.8%,发病主要与犬的免疫状况、年龄、品种有关、性别和季节等因素有关。     

Hemophagocytosis and Histoplasma‐like fungal infection in 32 cats

Histoplasmosis is one of the most common systemic fungal diseases in cats from the United States. It commonly causes respiratory or disseminated disease and is often associated with one or more cytopenias. Here, we describe 32 cats in which a Histoplasma‐like fungal infection was associated with concurrent hemophagia in at least one sample site, commonly spleen, bone marrow, liver, and/or lymph node. The degree of hemophagia was characterized as moderate or marked in the majority of cases, and in all cases, there was a predominance of phagocytized mature erythrocytes. A few cases also had macrophages with phagocytized erythroid precursors, platelets, and/or neutrophils. Complete blood count results were available for 25 cats, and cytopenias were common (20/25), including solitary anemia (10), anemia and thrombocytopenia (5), solitary neutropenia (2), pancytopenia (2), and anemia and neutropenia (1). Bone marrow samples were only available in a small subset of cases, preventing the further assessment of the causes of the cytopenias. Hemophagocytosis has been previously reported in cats with neoplastic diseases and a cat with calicivirus infection, and likely occurs with other conditions as well, such as hemorrhage or hemolysis. Results of this report suggest that systemic fungal disease is an additional differential to consider when there is hemophagia in a feline cytology sample.     

Diagnosis of congenital and adult-onset hypothyroidism in cats

Whereas hyperthyroidism is the most common endocrine disorder in the cat, hypothyroidism is the least common feline endocrine disorder. This is a the result of several factors including low index of suspicion, rarity of the naturally occurring hypothyroidism in cats, and a lack of species specific tests for endogenous TSH and antithyroglobulin antibodies. Nonetheless, hypothyroidism does occur in cats, especially in kittens and after radioactive treatment for hyperthyroidism. The clinician should become familiar with the common presentations of congenital and adult-onset hypothyroidism in cats. In addition, some of the tests specific to dogs (such as endogenous canine TSH) may be utilized to diagnose subclinical hypothyroidism in cats. Fortunately, the treatment of feline hypothyroidism with synthetic levothyroxine is both straightforward and effective.