Characterization of fowl adenoviruses from outbreaks of inclusion body hepatitis/hydropericardium syndrome in Chile.

Three fowl adenovirus (FAV) isolates (341, 344, and 215) obtained during 1996-97 from field outbreaks of inclusion body hepatitis/hydropericardium syndrome (IBH/HPS) affecting broilers and broiler breeders in Chile were characterized by virus neutralization tests (VNTs) and restriction enzyme analysis of a DNA fragment. Furthermore, the pathologic characteristics of one of these FAV isolates (FAV 341) was studied in experimentally infected chickens. The VNTs conducted with isolates 341 and 344 against reference strains and antisera belonging to each of 12 FAV serotypes demonstrated a close antigenic relationship with strain KR5 of the FAV serotype 4. Polymerase chain reaction using the primers H3/H4 and subsequent HpaII digestion was used for serotype identification of isolates 341 and 215. The length of the PCR products and the restriction profiles of isolates 341, 215, and the reference strain KR5 (FAV4) were identical. The present results confirmed the classification of all three isolates as FAV4. The pathogenicity test with 1000 mean tissue infectious dose of isolate 341 inoculated intramuscularly in 20-day-old specific-pathogen-free chickens resulted in the death of 9% (two birds) six days postinoculation (PI). Both birds showed characteristic IBH/HPS gross and microscopic lesions; the remaining birds, sacrificed at day 10 PI, showed less severe lesions. On the basis of epidemiologic and experimental data of the virulence of Chilean FAV isolates, and the pathogenicity results with isolate 341, we speculate that Chilean FAV strains may require an association with other agents (immunosuppressive agents) to induce IBH/HPS outbreaks in the field.     

Chicken anemia virus and fowl adenoviruses: association to induce the inclusion body hepatitis/ hydropericardium syndrome

The effects of a simultaneous and/or a subsequent coinfection with chicken anemia virus (CAV) isolate 10343 and fowl adenovirus (FAV) isolate 341 in specific-pathogen-free light chickens were evaluated. The simultaneous coinfection was conducted by the intramuscular route, whereas the subsequent coinfection trial considered FAVs administered orally. In trial 1, 20-day-old chickens simultaneously coinfected with CAV (10343) and FAV (341) intramuscularly (i.m.) showed 55% mortality and characteristic signs and lesions of inclusion body hepatitis/hydropericardium (IBH/HPS). In contrast, birds singly infected with FAV i.m. showed 10% mortality due to IBH/HPS. Trial 2 showed that birds receiving FAV 341 orally at day 7 post-CAV intramuscular infection (group A) developed a mild form of IBH/HPS with presence of inclusion bodies (INIBs) in 60% of the group and virus-neutralizing antibodies against FAV 341. Group B (FAV orally 14 days after CAV) showed significant decreased weight gain, nonspecific microscopic lesions in the liver, spleen, bursa, and thymus, and an antibody response against FAV 341. However, no INIBs could be detected in the hepatocytes of these chickens. Group C (FAV orally 35 days after CAV) showed nonspecific histopathologic changes in the liver and no antibody response to FAV. The oral single infection with FAV isolate 341 induced neither mortality nor macroscopic lesions of IBH/HPS in the birds. The present results corroborate previous reports on pathogenicity of Chilean FAV isolates, which suggest that synergism with other viruses or prior immunosuppression is necessary to produce IBH/HPS in chickens. These results also suggest that the susceptibility of chickens to FAV oral infection resulting in IBH/HPS varies throughout the course of CAV infection.     

Differentiation of avian poxvirus strains on the basis of nucleotide sequences of 4b gene fragment

Investigations for detection and differentiation of nine avian poxviruses (APVs) were carried out by the use of a polymerase chain reaction (PCR) combined with restriction enzyme analysis (REA) and further nucleotide sequence analysis. With one primer set, which framed a region within the fowl poxvirus 4b core protein gene, we were able to detect APV-specific DNA from 19 tested strains and isolates belonging to five defined Avipoxvirus species and four previously undefined isolated species. PCR results revealed no recognizable differences in size of amplified fragments among the different APVs. REA of PCR products with MseI and EcoRV allowed us to differentiate most of the tested avipox species. Nucleotide sequence analysis of the amplified fragments showed a nucleotide similarity of 72%-100% among the different species. Phylogenetic analysis documented five distinguishable sequence clusters in accordance with results obtained by REA. PCR in combination with REA and sequencing of the amplified fragments is a rapid and effective diagnostic system, and it is a new approach to refine epidemiologic studies of APV infections.     

FTA liver impressions as DNA template for detecting and genotyping fowl adenovirus

The feasibility of using liver impressions on Flinders Technology Associates (FTA filter paper for the collection, inactivation, and molecular analysis of fowl adenovirus (FAV) was evaluated. FAV I European Union (EU) serotype 1 spotted on FTA was shown to be inactivated using specific-pathogen-free (SPF) primary chicken embryo liver cell culture as indicated by absence of cytopathic effect. Sensitivity of the polymerase chain reaction (PCR) test using tenfold dilutions of allantoic fluid from 100 to 10-4 for the detection of adenovirus serotype 1 on FTA cards was determined to be 0.0005 mean tissue culture infectious dose per FTA spot. The stability of the DNA from liver impressions on the FTA was found to be 198 days when stored at -20 degrees C. In a trial, inclusion body hepatitis (IBH) was experimentally reproduced in SPF chickens inoculated with FAV I EU serogroup 1, 4, 8, or 11, which presented weakness, pallor, depression, dehydration, and mortality within 6 days after inoculation. PCR performed on FTA liver impressions from the inoculated birds was able to detect all four viruses, and the nucleotide sequence analysis of the amplified PCR products (1219 bp of the hexone gene) revealed the expected serotypes. In addition to the trial, 55 clinical samples were analyzed from liver impressions on FTA cards, and FAV was detected in 11 of 55 (20%). Sequencing analysis showed that the viruses were EU serotypes 4, 5, 9, and 10. The results demonstrate that FTA filter paper inactivates the FAV I and maintains the DNA template for molecular analysis.     

Epizootiology of fowls adenoviruses

Fowl adenoviruses of the serotype 4 from Germany were characterised by restriction enzyme analysis in comparison to isolates from Asia, South America and the FAV4 reference strain KR5. Only strain Da60 which was isolated from a psittacine aviary was identical with the reference strain KR5. None of the isolates was identical with the highly pathogenic strains from India and Ecuador. One-day-old chicks were infected orally and intramuscularly with the reference strain KR5, the psittacine isolate Da60 and isolate K1013 from Ecuador. Whereas no mortality was seen with the two strains KR5 and Da60, the mortality with K1013 was 100%. The main pathological signs were a swollen liver with necrosis and a lymphocyte depletion with a loss of the follicle structure. To investigate a second subject of avian adenovirus epizootiology several FAVs were characterized serologically and with PCR which was combined with the digestion of the PCR products. Including the reference strains, both methods were compared. It was shown that the digestion of the PCR products allows a clear attribution to a specific serotype, which underlines the usefulness of this method for diagnostic purposes.     

Establishment and Application of a Nested PCR Assay for Detection of Fowl Adenovirus Group Ⅰ

According to the sequence of hexon gene of fowl adenovirus groupⅠ(FAVⅠ) strain published in GenBank,two pairs of primers were designed and synthesized.The outer primers amplified a fragment of 475 bp in length, and the inner primers amplification fragment was 237 bp in length. A nested PCR assay for rapid detection of FAVⅠ was established.A specific 237 bp fragment was amplified from DNA templates of FAVⅠstrain,but no bands were amplified with templates extracted respectively from avian influenza virus (AIV) subtype H9,Newcastle disease virus (NDV), infectious bursal disease virus (IBDV),duck plague virus (DPV), reticuloendotheliosis (REV), avian reovirus (ARV), Marek's disease virus (MDV). Sensitivity of the 1st and 2nd amplifications by the nested PCR assay were 100 pg and 1 fg,respectively.The sensitivite of the 2nd amplifications increased by 10⁵ times.The results showed that the nested PCR was specific,sensitive,rapid,accurate,and could be used as a routine assay for the detection of FAVⅠ.This method had good reproducibility, specificity and sensitivity, and might detect low content FAVⅠ accurately and rapidly. This method could be used as a method for the diagnosis and detection of clinical cases,and molecular epidemiological investigation of FAVⅠ.     

鸭瘟病毒北京株UL6和UL7基因的克隆及序列测定

依据文献报道的鸭瘟病毒（DPV）的核苷酸序列，设计和合成了二对引物，分别为SP1和SP2，LP1和LP2。北京株鸭瘟病毒于鸭胚中增殖，差速离心和蔗糖密度梯度离心纯化，按酚－氯仿法抽提DNA。然后以DPV DNA为模板进行PCR，分别扩散增出与理论相符的421bp和1209bp的二个DNA片段，并将它们克隆于质粒pGEM-T Easy中。经酶切和质粒PCR，筛选含有目的基因的重组质粒。重组质粒以步移法从双方向测序，获1586bp的核苷酸序列。研究发现这1586bp的核苷酸序列与文献报道的UL6和UL7基因序列的同源性为99％，仅有一个碱基的差异。进一步作氨基酸分析，发现这个碱基所引起的变异为无意义突变（CCT→CCC）。结果表明，鸭瘟病毒的UL6和UL7基因在不同的毒株中高度保守。     

Identification of genotypes of Giardia intestinalis isolates from dogs in Japan by direct sequencing of the PCR amplified glutamate dehydrogenase gene

Giardia has been detected in domestic dogs in Japan, but the genotype of isolates has remained unclear because identification has relied on conventional microscopy. Here we tried to identify the genotypes of four isolates from dogs in Japan by direct sequencing of the PCR amplified Giardia glutamate dehydrogenase (GDH) gene. The primer pair GDHF3 and GDHB5, targeting the GDH gene, was designed to prime a region of the GDH gene sequence conserved in the strains found to have the dog-specific genotype. The specific PCR product (approximately 220 bp), amplified with this primer pair, was only observed when Giardia DNA was used as the template. The sequences of the diagnostic fragments were identical among the isolates from dogs, and were differed by 15 bp or 1 bp from the strains, which were found to be the dog-specific genotypes, Assemblage C or D respectively. To verify the identity of the amplified DNA, a phylogenetic analysis was performed. Consequently, the sequence of the isolates from dogs clearly clustered with the strain found to be Assemblage D with neighbor-joining analyses. Therefore, all the isolates from dogs examined were identified as the dog-specific genotype, Assemblage D. In the present study, we revealed the genotype of Giardia isolates in Japan, and showed that direct sequencing of the PCR product amplified with the primer pair GDHF3 and GDHB5 was a useful tool for distinguishing between the zoonotic and dog-specific genotypes.     

Vertical induction of the inclusion body hepatitis/hydropericardium syndrome with fowl adenovirus and chicken anemia virus

The hypothesis that fowl adenovirus (FAV) and chicken anemia virus (CAV), transmitted vertically and simultaneously, induce the inclusion body hepatitis (IBH)/hydropericardium (HP) syndrome in progeny chickens was tested. Thus, 35-wk-old light brown layer breeders, showing absence of antibodies against FAV and variable titers against CAV, were intramuscularly singly infected with the FAV serotype 4 isolate 341 or dually infected with CAV (isolate 10343) and FAV. All hens (groups A [FAV alone], B [FAV + CAV], and C [noninfected]) were clinically healthy throughout the experimental period. Both infectious viruses FAV and CAV were isolated from progenies obtained as early as 5 days after infection of their breeders. Hematocrit, serum proteins, and aspartate-aminotransferase values showed a few statistical differences between the progeny groups. Most of these differences were detected in the progeny chickens of group B. However, almost all values met reference values for the species. The pathologic findings showed that progeny chickens obtained from both singly and dually infected breeders developed macroscopic and histopathologic changes of IBH/HP. The pathologic findings shown by progeny chickens of group A (FAV) were not expected because neither synergism nor prior immunodepression by CAV was concurrent. Chickens of group B (CAV + FAV) also developed IBH/HP. Although not many differences in the evaluated parameters between groups A and B were statistically significant, most pathologic findings of group B indicated a more severe manifestation of the disease. However, because FAV alone did reproduce the syndrome, the results shown by group B would not allow a definitive confirmation of the hypothesis that the association of FAV and CAV is necessary for the successful induction of the IBH/HP syndrome in chickens when transmitted vertically.     

鹅细小病毒和番鸭细小病毒双重PCR检测方法的建立

根据GenBank上登录的鹅细小病毒(GPV)和番鸭细小病毒(MDPV)基因序列,分别设计合成针对GPV非结构蛋白(NS)和MDPV NS2-VP1基因片段的2对引物GPV U/L和MDPV U/L,将GPV和MDPV提取核酸混合后作为模板,优化PCR反应条件,建立了能同时检测这2种病毒的双重PCR。特异性试验结果显示,引物GPV U/L仅特异性扩增出GPV-GZ1和GPV-GZ2株730bp核酸片段,引物MDPVU/L仅特异性扩增出MDPV的624bp核酸片段,双重PCR扩增出长度分别为730bp和624bp的2条特异性片段,而扩增鸭瘟病毒(DPV)和鹅副黏病毒(GPMV)的核酸扩增结果均为阴性。敏感性试验结果显示,双重PCR能同时检测到14.4pg的GPV核酸和28.8pg的MDPV核酸。结果表明,建立的双重PCR可用于GPV和MDPV的鉴别诊断和联合检测。     

A multiplex polymerase chain reaction for discriminating Erysipelothrix rhusiopathiae from Erysipelothrix tonsillarum.

Erysipelothrix rhusiopathiae is the causative agent of swine erysipelas, and it causes great economic losses in Japan and worldwide. In meat inspection, it is very important to distinguish E. rhusiopathiae from other bacteria showing similar clinical signs of disease or similar bacterial characteristics. To distinguish E. rhusiopathiae from Erysipelothrix tonsillarum, 2 polymerase chain reaction (PCR) systems were combined. The primer sets ERY-1F and ERY-2R were designed to amplify 2210 base pairs (bp) of nucleotide sequence specific for E. rhusiopathiae chromosomal DNA, and the primer sets MO101 and ERS-1R were designed to amplify 719 bp of nucleotide sequence including a highly conserved region of genus Erysipelothrix 16S rRNA. Two fragments were amplified when E. rhusiopathiae was used as the PCR template using the primer sets, whereas a single fragment was amplified when E. tonsillarum was used as the template. No fragments were amplified when nucleic acid from other bacteria that cause clinical signs similar to swine erysipelas were used as the template. Moreover, 5 specimens collected from postinspected swine carcasses were diagnosed as E. rhusiopathiae using the PCR described in this study, in agreement with results of microbiological tests for the genus Erysipelothrix, whereas negative samples were negative both in conventional bacterial tests and by PCR. The detection limit of multiplex PCR ranged from 10(2) to 10(4) colony forming units per reaction tube for positive samples. These results suggest that this method is useful for screening of swine erysipelas in meat inspection centers.     

天祝白牦牛ANGPTL4基因的单核苷酸多态性研究

[目的]本研究旨在对牛ANGPTL4基因进行SNPs检测,并研究其与天祝白牦牛部分经济性状的相关性。[方法]以36头天祝白牦牛为研究对象,采集其血样并提取基因组DNA,设计ANGPTL4基因的特异引物对P1和P2,对ANGPTL4基因的部分序列进行扩增,并利用PCR-SSCP结合测序的方法检测扩增产物序列的多态性。[结果]以自行设计的上述两对引物扩增分别获得长为260 bp（位于GenBank公布序列NC-007305.2的296~555 bp间）和170 bp（位于序列NC-007305.2的4 610~4 779 bp间）的天祝白牦牛ANGPTL4基因的两个片段。引物对P1的扩增产物经变性聚丙烯酰胺凝胶电泳,出现两种带型：AA和AB。AA和AB两种基因型频率分别为0.9444和0.0556。多态信息含量是0.1000,为低度多态。对具有不同带型的个体测序结果表明,引物对P1的扩增片段上有两处发生碱基突变,分别位于ANGPTL4基因的第466 bp处（T→C）和479 bp处（T→C）,导致相应的氨基酸改变Cys→Arg,Phe→Ser。在引物对P2的扩增片段上没有发现SNPs位点。[结论]引物对P1的扩增位点可以尝试作为天祝白牦牛品质改良的辅助选择标记。     

Development and evaluation of a polymerase chain reaction assay using the 16S rRNA gene for detection of Eperythrozoon suis infection.

The 16S ribosomal RNA (rRNA) gene of Eperythrozoon suis was amplified using gene-specific primers developed from GenBank sequence accession U88565. The gene was subsequently cloned and sequenced. Based on these sequence data, 3 sets of E. suis-specific primers were designed. These primers selectively amplified 1394, 690, and 839 base-pair (bp) fragments of the 16S rRNA gene from DNA of E. suis extracted from the blood of an experimentally infected pig during a parasitemic episode. No polymerase chain reaction (PCR) products were amplified from purified DNA of Haemobartonella felis, Mycoplasma genitalium, or Bartonella bacilliformis using 2 of these primer sets. When the primer set amplifying the 690-bp fragment was used, faint bands were observed with H. felis as the target DNA. No PCR products were amplified from DNA that had been extracted from the blood of a noninfected pig or using PCR reagents without target DNA. The detection limits for E. suis by competitive quantitative PCR were estimated to range from 57 and 800 organisms/assay. This is the first report of the utility of PCR-facilitated diagnosis and quantitation of E. suis based on the 16S rRNA gene. The PCR method developed will be useful in monitoring the progression and significance of E. suis in the disease process in the pig.     

Development of PCR-based tests for the identification of North American isolates of epizootic haemorrhagic disease virus.

A serogroup-specific polymerase chain reaction (PCR) assay and isolate identification strategies (restriction endonuclease analysis (REA) and nucleotide sequencing) were developed for the detection of North American isolates of epizootic haemorrhagic disease virus (EHDV). PCR primers (EHDV-pr4, EHDV-pr5) were designed to hybridize to the L3 gene of a North American isolate of EHDV serotype 1. Total nucleic acid was extracted from preparations of infected tissue culture and PCR was performed using a cDNA-PCR kit, according to the manufacturer's specifications. The PCR assay generated a 459 base pair product from North American isolates of EHDV serotypes 1 and 2, while bluetongue virus (BTV) serotypes 10, 11, 13, and 17, and cell controls, failed to demonstrate PCR products. Slight modifications allowed for the PCR detection of EHDV-1 and -2 in white-tailed deer blood (Odocoileus virginiatus); PCR fragments were not amplified from uninfected deer blood. A number of restriction endonucleases and sequencing primers were evaluated for their utility in isolate identification experiments. Specifically, REA employing HincII and cycle sequencing with an internal primer (EHDV-1-pr3) proved most successful for identifying isolate-specific genome markers. The techniques presented are expected to prove valuable for rapid and specific detection of possible future EHDV incursions in wild and domestic animal species.     

Development of a multiplex PCR and PCR-RFLP method for serotyping of Avibacterium paragallinarum

Avibacterium (Haemophilus) paragallinarum (A. paragallinarum) is a causative agent of infectious coryza in chickens and is classified into three serovars by agglutination tests. In an effort to identify the serovars easily, PCR and PCR-RFLP were employed. As the target gene for PCR, the hypervariable region of HMTp210, which encodes the HA antigen, was used. PCR using primer sets around the hypervariable region amplified 0.8, 1.1 and 1.6 kbp fragments for serovars A, B and C, respectively. Alternatively, the 1.6 kbp fragments were amplified with another primer pair encompassing the hypervariable region and was subjected to digestion with Bgl II, which resulted in the detection of serovar-specific digestion patterns. These results indicate that PCR and PCR-RFLP using the hypervariable region of HMTp210 are alternative methods to identify the serovar of A. paragallinarum.     

Different DNA methylation patterns detected by the Amplified Methylation Polymorphism Polymerase Chain Reaction (AMP PCR) technique among various cell types of bulls

Background

The purpose of this study was to apply an arbitrarily primed methylation sensitive polymerase chain reaction (PCR) assay called Amplified Methylation Polymorphism Polymerase Chain Reaction (AMP PCR) to investigate the methylation profiles of somatic and germ cells obtained from Holstein bulls.

Methods

Genomic DNA was extracted from sperm, leukocytes and fibroblasts obtained from three bulls and digested with a methylation sensitive endonuclease (HpaII). The native genomic and enzyme treated DNA samples were used as templates in an arbitrarily primed-PCR assay with 30 sets of single short oligonucleotide primer. The PCR products were separated on silver stained denaturing polyacrylamide gels. Three types of PCR markers; digestion resistant-, digestion sensitive-, and digestion dependent markers, were analyzed based on the presence/absence polymorphism of the markers between the two templates.

Results

Approximately 1,000 PCR markers per sample were produced from 27 sets of primer and most of them (>90%) were digestion resistant markers. The highest percentage of digestion resistant markers was found in leukocytic DNA (94.8%) and the lowest in fibroblastic DNA (92.3%, P ≤ 0.05). Spermatozoa contained a higher number of digestion sensitive markers when compared with the others (3.6% vs. 2.2% and 2.6% in leukocytes and fibroblasts respectively, P ≤ 0.05).

Conclusions

The powerfulness of the AMP PCR assay was the generation of methylation-associated markers without any prior knowledge of the genomic sequence. The data obtained from different primers provided an overview of genome wide DNA methylation content in different cell types. By using this technique, we found that DNA methylation profile is tissue-specific. Male germ cells were hypomethylated at the HpaII locations when compared with somatic cells, while the chromatin of the well-characterized somatic cells was heavily methylated when compared with that of the versatile somatic cells.     

A detection assay forCampylobacter fetus in bovine semen by restriction analysis of PCR amplified DNA

A rapid screening assay forCampylobacter fetus in bull semen was developed using the polymerase chain reaction (PCR) and restriction endonuclease analysis (REA) to complement isolation by culture. An oligonucleotide primer pair (C₁/C₂) from the hypervariable region of 16S rRNA ofC. fetus was used to amplify a 362 base pair fragment by PCR. The PCR/REA assay, which is completed in 10 hours, detected as few as threeC. fetus subsp.venerealis cells in experimentally infected raw bull semen and in semen diluted with milk or egg yolk Tris (EYT). All the strains tested, of both subspecies ofC. fetus, were amplified, as were some otherCampylobacter species. Restricting the amplified products byAluI differentiatedC. fetus from the other organisms. There was no visible product generated by PCR fromC. sputorum subsp.bubulus, a saprophytic organism found in the prepuce of bulls, or from seven other species of bacteria found in semen. A modification of the PCR assay, using another primer pair (C₃/C₂) and two temperature PCR cycling conditions, increased the probability of detectingC. fetus subsp.venerealis. PCR amplification followed by REA could be used to screen bovine semen rapidly forC. fetus. In most cases, sequencing of C₁/C₂ PCR generated products would be preferable for distinguishing between the two subspecies ofC. fetus.Abbreviations AI artificial insemination - bp base pair - Cff Campylobacter fetus subsp.fetus - Cfv Campylobacter fetus subsp.venerealis - EYT egg yolk Tris - MH Mueller-Hinton - PCR polymerase chain reaction - REA restriction endonuclease analysis - TE Tris-EDTA     

Identification of Flavobacterium and Flexibacter Species by Species-Specific Polymerase Chain Reaction Primers to the 16S Ribosomal RNA Gene

Abstract

Species-specific polymerase chain reaction (PCR) primers have been developed for the identification of the causative agents of warmwater and marine finrot in fish: Flavobacterium columnare (Flexibacter columnaris) and Flexibacter maritimus. Differences in gene sequence in the bacterial small-subunit (16S) ribosomal RNA (rRNA) were used to design the species-specific PCR primers. The previously reported species-specific PCR primers Psy1 and Psy2 for the identification of Flavobacterium psychrophilum (Flexibacter psychrophila), the causative agent of coldwater finrot, were also used to develop a speciation scheme for all three bacterial finrots. These three primer sets were successful in discriminating among yellow-pigmented bacteria as well as in speciating the three major pathogenic flexibacteria to fish. The primer sets were designed to produce uniquely sized subproducts of 16S rRNA for each species: Flavobacterium psychrophilum (1,100 base pairs, bp), F. columnare (800 bp), and Flexibacter maritimus (400 bp). These primers were shown to correctly speciate field isolates in double-blind experiments (P = 0.01).     

The high prevalence of Helicobacter sp. in porcine pyloric mucosa and its histopathological and molecular characteristics

This study examined the prevalence of Helicobacter infection in the pyloric mucosa of pigs and its histopathological and molecular characteristics. Forty porcine pyloric samples were examined for Helicobacter infection by silver staining and PCR assay. The PCR product (376 bp) was digested with NdeII to differentiate between Helicobacter heilmannii and Helicobacter pylori. Another PCR assay run to produce an 1157 bp fragment was performed using a primer set designed from the 16S rRNA gene of Candidatus H. suis, and its product was cloned and sequenced. Infection rates were 62.5% (25/40) and 95.0% (38/40) as determined by silver staining and the PCR assay, respectively. On histopathological examination, lymphoid follicle aggregation in the pyloric mucosa and granulocytic migration into the lumen of pyloric glands were observed in 24 (60.0%) and 33 (82.5%) gastric samples, respectively. All PCR products, except that of H. pylori, were cut into two fragments of 147 and 229 bp by enzymatic digestion with NdeII. Sequencing of the 16S rRNA gene showed that the bacterium had 99.57% (1152 bp/1157 bp) homology to the 16S rRNA gene of Candidatus H. suis.     

PCR test for detecting Taenia solium cysticercosis in pig carcasses

Polymerase chain reaction (PCR) test was employed to detect Taenia solium DNA in muscle lesions for validation of the meat inspection results of slaughtered pigs. Two sets of oligonucleotide primers, one targeted against the large subunit rRNA gene (TBR primers) and the other targeted against cytochrome c oxidase subunit 1 gene (Cox1 primers) of T. solium were used in this study. On reactivity in PCR test, the TBR primers and the Cox1 primers yielded products of 286 and 984 bp, respectively, in cysticercosis positive cases. Both the sets of primers were found to be highly specific, since they did not yield any PCR product in negative controls. A total of 225 pig carcasses were screened for cysticercosis by meat inspection, out of which 25 carcasses with visible cysts (16 viable and 9 degenerated cysts) were also confirmed to be positive for cysticercosis in PCR test. However, out of the 35 carcasses with suspected lesions on meat inspection, only two were found to be positive for cysticercosis in PCR test. The detection limits for both the primer sets were analyzed. The TBR primer set could detect up to 10 pg of cysticercus DNA, whereas the Cox1 primer set could detect only up to 1 ng. It is evident from the study that PCR test is an efficient tool for validation of meat inspection results and also to rule out ambiguity in carcass judgment of suspected cases of porcine cysticercosis.