Serodiagnosis of Fasciola gigantica infection in buffaloes

The agar gel precipitation test (AGPT), counter immunoelectrophoresis (CIEP) and indirect haemagglutination (IHA) were evaluated for the diagnosis of fascioliasis due to Fasciola gigantica in buffaloes. The sensitivity of these tests varied with the intensity of infection; and was greatest when the fluke burden in liver exceeded 100. CIEP detected 76.06% of infected sera and was most sensitive, followed by IHA which detected 68.37% of the infected sera. The AGPT was found to be least sensitive, detecting only 57.4% of the infected sera. Although these tests were limited by the occurrence of false-positive reactions, their use may be an aid for effective diagnosis of fascioliasis in buffaloes.     

Detection of avian adenovirus by polymerase chain reaction

An avian adenovirus-specific polymerase chain reaction was developed. The origin of primers was from the DNA sequence data of the chicken embryo lethal orphan avian adenovirus virus genome. An avian adenovirus-specific 421-bp DNA product was amplified by these primers from group I of adenovirus containing 12 serotypes and serotypes of adenovirus from group II and group III. The adenovirus-specific DNA product was also amplified from the 19 field isolates of avian adenoviruses but not from the mammalian adenovirus and other avian pathogenic viruses and bacteria. As little as 1 fg of avian adenovirus DNA was detected by gel electrophoresis and Southern blot analysis.     

Detection of pigeon circovirus by polymerase chain reaction

Pigeon circovirus was identified by polymerase chain reaction (PCR) in young pigeons belonging to 12 different lofts. Viral DNA was extracted from formalin-fed, paraffin-imbedded tissues containing primarily bursa and occasionally liver and spleen with a commercial kit. PCR primers were selected from a published sequence for columbid circovirus and evaluated in a PCR assay. The histopathologic examination of various tissues revealed basophilic globular intracytoplasmic inclusions in the mononuclear cells of the bursa of Fabricius and occasionally in the spleen characteristic for a circovirus. Transmission electron microscopy of a few bursas of Fabricius revealed virus particles measuring 18-21 nm. All the samples were negative by PCR for psittacine beak and feather disease (PBFD) virus and chicken infectious anemia virus. The primers for both pigeon circovirus and PBFD virus did not react in PCR with the chicken anemia virus DNA. Most of the circovirus-infected pigeons had concurrent infections of Escherichia coli, Salmonella, Pasteurella, Aspergillus, candidiasis, nematodiasis, or capillariasis.     

片形吸虫DNA随机扩增多态性分析

为区别从南京市江宁县采集的片形吸虫非典型形态虫体，应用随机扩增多态性DNA（RAPD）技术，对6株片形吸虫总DNA进行了扩增。结果，10条引物中有8条能产生扩增图谱，电泳图谱经聚类分析，与传统的分类结果一致，并表明来自江宁的片形吸虫既有形态典型的肝片形吸虫，也有形态不典型的大片形吸虫。     

Detection of avian polyomavirus infection by polymerase chain reaction using formalin-fixed, paraffin-embedded tissues

Avian polyomavirus infection in psittacines was diagnosed in tissues by the use of polymerase chain reaction (PCR) test. The tissues used in the procedure were either formalin-fixed tissues embedded in paraffin blocks or fresh tissues (heart, liver, and spleen) collected from the psittacines during necropsy. DNA was extracted from these tissues and was tested with the published primers for avian polyomavirus VP1 gene in the PCR that yielded an amplicon of 550 base pair size, which was then visualized by electrophoresis. The amplicon size was consistent with avian polyomavirus. The PCR test was found to be an effective method for identifying avian polyomavirus infection in both formalin-fixed, paraffin-embedded and fresh tissues from psittacine birds of different age groups.     

Detection of persistent Cytauxzoon felis infection by polymerase chain reaction in three asymptomatic domestic cats

Repeated polymerase chain reaction (PCR) testing of 3 asymptomatic domestic cats were positive for Cytauxzoon felis DNA, suggesting persistent infection. Two cats initially presented with clinical signs consistent with acute cytauxzoonosis and, in both cases, signs of illness resolved after treatment. Parasitemia was detected in peripheral blood smears from these cats upon presentation with illness and, at subsequent follow-up appointments, in the absence of clinical illness. Polymerase chain reaction analysis was positive for C. felis from blood sampled at each time point. A third cat, a housemate of a cat fatally infected with C. felis, was preventatively treated for infection at the time of the housemate cat's death. This contact cat, having never shown signs of clinical illness consistent with cytauxzoonosis infection, had no detectable parasitemia but was positive for C. felis on repeated PCR testing. Detection of asymptomatically infected cats allows for the possibility of a yet unrecognized population of infected domestic cats that may have the capacity to serve as an additional reservoir host for C. felis, altering the currently accepted paradigm of C. felis transmission to domestic cats through bobcats as the reservoir host. In cases of very low parasitemia, more sensitive means of parasite detection, such as PCR testing, may be necessary to detect infected cats. Increased detection of asymptomatically infected cats will aid in understanding the epidemiology of C. felis infection and enhance the ability to prevent this highly fatal infectious disease of domestic cats.     

Detection of leptospires in bovine semen by polymerase chain reaction

OBJECTIVE: In view of the considerable importance of venereal transmission of bovine leptospirosis, the objective of the present study was to compare the polymerase chain reaction (PCR), culture/isolation and serology to detect leptospire infection in bovine semen. DESIGN: Blood for serologic examination and semen for bacterial culture and PCR were collected from 20 bulls at artificial insemination centres in Brazil. Each animal was sampled twice for serology. RESULT: Forty-five percent (9/20) of the serum samples collected showed agglutinin titers to serovar hardjo in the first sample and 25% (5/20) had agglutinin titers to serovar hardjo in the second sample. Eighty percent (16/20) of semen samples were positive by PCR. Leptospires could not be isolated from any of the semen samples examined. CONCLUSION: Polymerase chain reaction can be a method of great potential for the detection of leptospires at artificial insemination centres.     

副溶血性弧菌PCR检测方法的建立

为了建立适于基层单位应用的针对副溶血性弧菌检测的特异PCR方法,检测副溶血性弧菌的基因序列.针对该菌的属特异性基因t1基因进行引物设计,扩增片段大小为449 bp.运用该PCR法可特异性的从副溶血性弧菌ATCC17802标准株中扩增出目的基因片段,与GenBank上发表的序列同源性为100%.而经常污染海产品的溶藻弧菌,嗜水气单胞茵标准株J-1,铜绿假单胞茵标准株ATCC27853结果均为阴性.该PCR法最低检出菌体DNA量为10-2ng以及最低检出菌数为5.7 × 103 CFU/mL.对临床上送检的35份样品进行菌体DNA PCR方法检测并同时与国标GB/T4789.7-2003中使用的检测方法进行比对,两种检测方法的结果完全符合,与国标中使用的检测方法相比可大大缩短鉴定时间.因此该PCR法能快速鉴定当前副溶血性弧菌流行群,有利于流行病学溯源等研究.     

应用PCR技术检测鹅细小病毒

根据鹅细小病毒GPVH1株结构蛋白VP1与VP3编码基因非重叠核苷酸序列设计了一对引物GPR1 /GPR2 ,用这对引物对感染器官内的GPR和接种鹅胚增殖的GPV进行PCR扩增 ,其结果引物GRP1 /GPR2能特异性地扩增GPVVP1 VP3区段核苷酸序列 ,扩增出与设计核苷酸片段大小相符的 0 6kb的序列 ,而对照的鹅副粘病毒cDNA及鸭瘟病毒 (DPR)DNA对照组均出现阴性结果。     

Protection against Fasciola gigantica infection in rats administered metacercarial antigens

Immature worms were recovered from the liver parenchyma of rats which received extracts of metacercariae or adult worms of Fasciola gigantica, after challenge infection. When resistance was assessed by counting the number of immature worms, exposure of the animals to extracts of these two stages of the flukes conferred a significant degree of protection against challenge with F gigantica metacercariae. Rats given the extract of metacercariae were more resistant to infection than rats immunised with the extract of adult worms.     

Detection of feline coronavirus infection in captive cheetahs (Acinonyx jubatus) by polymerase chain reaction.

Feline coronavirus genetic elements were detected by polymerase chain reaction from blood, fecal samples, and effusive fluid collected from 33 cheetahs in the U.S.A. Feline coronavirus-specific serum antibodies were also measured by indirect immunofluorescence. Ten cheetahs were positive for viral shedding by polymerase chain reaction, whereas 13 were seropositive by immunofluorescence. Results of serology did not consistently correlate with shedding of virus, and the capture antigen used for detection of feline coronavirus-specific antibodies had a significant impact on results. Testing of samples from one population over a 1-yr period indicated chronic infection in some animals. These relatively healthy carrier animals were a source of virus for contact animals. Screening programs in cheetah populations for feline coronavirus infection may be most reliable if a combination of serologic analysis and viral detection by polymerase chain reaction is used.     

Tissue reaction to Fasciola gigantica in rabbits after treatment with rafoxanide

The tissue reaction to immature Fasciola gigantica was investigated in rabbits experimentally infected with 60 or 100 metacercariae. The rabbits were killed 1, 2, 4, 7, and 14 days after treatment, or 14 days after infection.The histopathological changes of the liver were similar in rabbits killed 1 or 2 days after treatment to those in untreated control rabbits. Dead immature flukes were recognized in the hepatic parenchyma 4, 7, and 14 days after treatment. During the early stage of infection they were surrounded by necrotic liver cells and eosinophils. The intensity of the reaction increased gradually and two distinct zones developed. The inner zone was composed of necrotic liver cells and eosinophils, and the outer zone was composed mainly of phagocytic mononuclear cells and fibroblasts.     

Host responses during experimental infection with Fasciola gigantica and Fasciola hepatica in Merino sheep II. Development of a predictive index for Fasciola gigantica worm burden

This study reports on the predictive relationship between serological, immunological and pathological responses following experimental inoculation with incremental doses of Fasciola gigantica in sheep. Fifty, 6-month-old, naive Merino wethers were allocated to one of 5 experimental groups, four of which received 50, 125, 225 and 400 metacercariae, respectively, whilst a 5th group acted as non-inoculated control. Strong individual correlations were observed between liver score, GLDH (glutamate dehydrogenase), GGT (gamma glutamyl transferase), CatL5 (cathepsin L5) antibody titre (IgG1, IgA), eosinophilia, and the total worm count or worm biomass. A combination of multiple indicator traits performed significantly better than any single indicator trait alone. The best predictive index accounted for up to 88% of observed worm burden (Wb) if information on inoculation dose was available. Without knowledge of inoculation dose, such as under field conditions, up to 67% of variation in worm burden could be predicted. In contrast, the best single predictor variable (liver damage score) accounted for up to 50% of worm burden, and in the absence of post-slaughter information, serum levels of anti-cathepsin IgA antibody titres accounted for 35% of predicted variation in worm burden. The utility of a predictive index under both field and experimental inoculation conditions is discussed.     

Detection of Ehrlichia canis by polymerase chain reaction in dogs from Portugal

Antibodies against Ehrlichia canis, the cause of canine monocytic ehrlichiosis, have been reported previously in clinically ill and stray dogs from Portugal. In this study, the 16S rRNA gene of E. canis was detected by the polymerase chain reaction (PCR) in 12/55 (22%) of dogs with suspected tick-borne disease in the Algarve region in Portugal.     

Primary experimental infection of riverine buffaloes with Fasciola gigantica.

The clinical course of the primary experimental Fasciola gigantica infection was investigated in riverine buffalo calves of the Murrah breed. Nine male calves aged 12-15 months were randomly assigned to two groups of five (Group I) and four (Group II) animals. Each animal in Group I, was orally infected with 1000 metacercariae (mc) of F. gigantica, whereas Group II animals did not receive any infection dose and served as uninfected controls. No clinical signs of fasciolosis were observed until the sixth week post-infection (PI). Group I animals, however, developed recognised symptoms of acute fasciolosis, comprising apyrexic inappetance, anemia, poor weight gain, diarrhoea and sub-mandibular and facial oedema, respectively, from 5, 6, 8, 16 and 17 weeks PI. The signs were intermittent in nature and of variable duration. The prepatent period was of 92-97 days (mean 95.2 +/- 3.1). One of the five infected animals died on Day 147 PI. At necropsy, 36.8 +/- 11.0% of the infection dose was recovered as adult fluke population. The gross lesions were primarily biliary in nature. Group II, the uninfected controls, throughout the study period of 165 days PI, did not show any symptom and were negative for F. gigantica. The study demonstrated that the onset of adverse effects of F. gigantica on the growth and health of the infected host was mainly noted during late prepatency much before coprological prediction and diagnosis. The significance of preventive therapy against fasciolosis during prepatency has been stressed in endemic areas.     

Detection of camel pox and vaccinia viruses by polymerase chain reaction

PCR following two methods of DNA extraction was used to confirm the growth of camel pox virus (CPV) and vaccinia virus in cell culture and chorioallantoic membrane. Results were compared with the commonly used neutralization test. The first method of DNA extraction was accomplished by using viral DNA in tissue culture supernatant and Chorioallantoic membrane, which was released by initial heating for 15 min at 99°C followed by ordinary PCR. In the second method DNA was extracted by using DNA Isolation Kit from tissue culture supernatant and used as a template. Rapid identification and differentiation of CPV and Vaccinia virus were achieved by PCR and this assay proved to be fast and feasible, and can be an alternative to orthodox serological methods.     

Cathepsin L cysteine proteinase in the diagnosis of bovine Fasciola gigantica infection

Cathepsin L cysteine proteinase from Fasciola gigantica was evaluated for its potential in the early prepatent detection of this helminth infection in bovine calves. Five cross-bred bovine calves were experimentally infected with 400 metacercariae/calf and evaluated for anti-cathepsin L antibody response. F. gigantica infection in these calves could be detected 4 weeks post-infection using an ELISA, dipstick ELISA and Western blotting with 100% sensitivity. The antigen was also used to detect F. gigantica field infection in cattle, by screening 256 sera of these animals by an ELISA, which demonstrated an overall infection rate of 26.95%. Preliminary studies showed that F. gigantica cathepsin L cysteine proteinase does not cross-react with Paramphistomum epiclitum, Gigantocotyle explanatum and hydatid cyst antigens. However, extensive studies on the cross-reactivity of this antigen with related helminth parasites of cattle and buffaloes are required, before this antigen can be considered suitable for immuno-diagnosis of fasciolosis in these ruminants.