Evaluation of colorimetric pH strips for wild animal meat using image analysis

Colorimetric pH strips (MColorpHast™) are very convenient to use, but the decision of pH is based on an individual's color perception and is, therefore, subjective. We developed a pH calculation program for the image of coloring strips on CIE1976 (L*a*b* color space), which slightly underestimated human judgment as the color of pH darker. This image analysis and three individuals' judgments were used for evaluating the strip's features for various qualities of meat from wild animals, and the results were compared with the assessments based on potentiometric pH. In both methods, dipping the strips in distilled water just before use improved the regression coefficient compared with that mentioned in the manual. The image analysis showed higher correlation than human judgments but slightly underestimate pH by a maximum of 0.13 unit from the regression line of the potentiometric pH. In addition, the image analysis revealed meat pigment changed pH higher on the color scale in the lower meat pH region. The strips must be used according to the manual, but dipping is effective when the meat surface is dry, and keeping the strips from touching the meat drip is important in lower pH region because the pigment affects the color of pH.     

A multiplex PCR assay for differentiating economically important gastrointestinal nematodes of cattle

A multiplex polymerase chain reaction (PCR) test was developed for identifying gastrointestinal (GI) nematodes that commonly infect cattle. This assay was developed using adult-derived genomic DNA and shown capable of discriminating parasite eggs from the feces of experimentally-infected animals at both the species and genus levels. Sequence data from internal (ITS) and external (ETS) transcribed spacers of the ribosomal DNA (rDNA) repeats as well as the 3'-end of the small subunit rDNA and 5'-end of the large subunit rDNA were used to generate five primer sets which, when used simultaneously in a multiplex PCR, produce a unique electrophoretic DNA banding pattern characterized by a single DNA fragment for Ostertagia ostertagi (257bp), Haemonchus placei (176bp), Oesophagostomum radiatum (329bp), Trichostrongylus colubriformis (243bp) and Cooperia oncophora (151bp). In a similar manner, the constructed primer sets amplified DNA from Ostertagia lyrata, Haemonchus contortus, Trichostrongylus axei, Cooperia surnabada and Cooperia punctata. With respect to H. contortus, a closely migrating doublet was generated suggesting size heterogeneity in the ETS which is consistent with multiple rDNA repeat units within this species. PCR analyses using mixtures of monospecifically-purified nematode eggs indicated a sensitivity of less than 0.5 egg-DNA equivalent per species. Although, not designed as a quantitative technique, relative PCR signal intensities corresponded to relative egg burdens within the DNA samples from mixed species of eggs.     

鸡毒支原体强弱毒株荧光定量PCR鉴别检测方法的建立

为建立一种能鉴别鸡毒支原体(MG)强、弱毒株的快速检测方法,本研究根据GenBank中MG强毒株和弱毒株的基因组序列,选取特异性保守区序列设计了2对引物和2奈探针,分别用于强弱毒株和弱毒株的检测,优化反应条件,建立了能区分MG强、弱毒株的荧光定量PCR检测方法.该法特异性强,对鸡常见呼吸道病原体的反应均为阴性;灵敏度高,可检测到100拷贝/μL的模板;稳定性好,批内和批间试验Ct值的变异系数小.本研究建立的MG强、弱毒鉴别检测方法简便、快捷,为该病的防控与净化提供新方法、新思路.     

猪巨细胞病毒PCR诊断技术的建立

猪巨细胞病毒(porcine cytomegalovirus,PC-MV)属于疱疹病毒科、β疱疹病毒亚科、巨细胞病毒属的成员;近来有人认为将本病毒划在玫瑰疹病毒属中更为恰当.     

胸膜肺炎放线杆菌血清学分型多重PCR方法的建立

10株胸膜肺炎放线杆菌参考菌株,经过纯化、扩增、提取基因组DNA,设计一套特异性引物,运用多重PCR技术,对外毒素基因apxⅠ、apxⅡ和apxⅢ进行扩增,产物经1.6%琼脂糖凝胶电泳,可将10株参考菌株分为5个血清群。血清Ⅰ群由259(血清1型)、263(血清5型)、267(血清9型)组成。血清Ⅱ群由260(血清2型)、262(血清4型)、264(血清6型)、266(血清8型)组成。血清Ⅲ群、血清Ⅳ群和血清Ⅴ群分别由261(血清3型)、265(血清7型)和268(血清10型)单独组成。对于血清Ⅰ群和血清Ⅱ群,通过apxⅣ进行扩增,结果259(血清1型)、263(血清5型)和267(血清9型)分别扩出2.4kb、2.8kb和1.6kb片段;262(血清4型)、264(血清6型)、260(血清2型)或266(血清8型)分别扩出1.6kb、2.0kb、2.8kb片段。     

鸡白痢沙门菌等位基因特异性PCR检测方法的建立

根据鸡白痢沙门菌与鸡伤寒沙门菌的rfbS基因在第237和第598位碱基的不同，设计和合成了等位基因特异性PCR引物，建立了快速检测鸡白痢沙门菌的PCR方法，并应用该方法对鸡白痢沙门菌临床分离样品进行了PCR鉴定。结果显示，该PCR方法能够特异性地鉴定鸡白痢沙门菌，检测灵敏度达100PgDNA。对35个经常规方法鉴定的鸡白痢沙门菌分离株应用等位基因特异性PCR方法进行鉴定，鉴定出33株鸡白痢沙门菌，符合率为94．3％。表明，建立的等位基因特异性PCR方法能够准确而快速地鉴定鸡白痢沙门菌。     

Feed resource base for scavenging village chickens in Sri Lanka

Summary The productivity of a population of scavenging village chickens in Sri Lanka has been assessed, and the scavenging feed resource base has been measured and analysed. The laying period lasted 34 ± 13 days and the batch size was about 20 eggs. The households ate 71% of the egg production. The mean egg weight was 48 g and the mean size of a set of eggs was 9·4. The hatching percentage was 67 ± 32 and the liveweight at 70 days averaged 313 g with a range of 142 to 492, by which time 65% of the chicks hatched had died. The age at first lay averaged 211 days when the pullets weighed 1,160 g. The broody period lasted from 3 weeks to 4 months depending on whether the hen hatched eggs, and for how long she tended the brood. The laying hens were actively scavenging for most of the daylight hours. The average amount of scavenged feed per household flock per day was 550 g dry weight with a proximate composition of 9·4% crude protein, 9·2% ether extract and 5·4% crude fibre. More than 70% of the feed intake was household refuse (27% cooked rice, 30% coconut residue, 8% broken rice and 36% other scraps). The remainder was from the environment (13% grass shoots, 8% small metazoans and 7% paddy rice). Proximate analyses of crop contents, household refuse and its major components were carried out. Dietary Ca and P levels were low in the village, as were plasma levels of these minerals. On a balanced commercial diet plasma Ca was still lower than that of hybrid commercial chickens. Suggestions are made for improving the productivity of the scavenging system with no requirement for inputs, and with inputs.

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Recurso Base Para Pollos Escarbadores De Villorrio En Sri Lanka

Resumen Se estudió la productividad de una población de pollos escarbadores en Sri Lanka y la base alimenticia se analizó. El período de postura duró 34 ± 13 dias y el grupo de unidades fue de aproximadamente 20 huevos. El consumo familiar fue de 71% de la producción. El peso promedio de las unidades fue de 48 g y el tamaño promedio de un juego de huevos fue 9·4. El porcentaje de eclosión de los mismos fue de 67 ± 32 y el peso vivo a los 70 días promedió 313 g con valores extremos de 142 a 492, con un porcentaje de mortalidad a esa edad de 65%. Le edad de postura promedió 211 dias, cuando las pollonas pesaron 1160 g. El periodo de empolle fluctuó entre 3 semanas y 4 meses dependiendo de si había enclosión de huevos, o de si la gallina (s) se encluecaban. Las gallinas escarbaban constantemente durante las horas de la mañana y tarde. El promedio de alimento tomado por grupo familiar de aves fue de 550 g peso seco con una composición aproximada de 9·4% de proteina cruda, 9·2% extracto de ether y 5·4% de fibra cruda. Más de 70% de la ingestion de alimento provino de desechos de cocina (27% arroz cocinado, 30% residuos de coco, 8% arroz partido y 36% otros residuos). El resto provino de pasto, metazoas, y 7% arroz pady. El análisis reveló niveles bajos de Ca y P en el alimento y en el plasma. Bajo una dieta comercial balanceada, los niveles de calcio plasmético fueron todavia bajos, en comparación a aquellos de hibridos comerciales. Se hacen recomendaciones para mejorar la productividad de estas aves criollas con insumos y sin ellos.

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Base Des Ressources Alimentaires Des Poulets Villageois Eleves Sur Dechets Menagers Au Sri Lanka

Résumé La productivité d'une population de volailles villaqueoises se nourrissant sur les déchets ménagers au Sri Lanka a été déterminée. La base de leurs ressources alimentaires à partir de ces déchets a été mesurée et analysée. La période de ponte a duré 34 ± 13 jours et le volume a été de l'ordre de 20 oeufs. Les propriétaires ont consommé 71 p. 100 de cette production. Le poids moyen des oeufs était de 48 q et leur dimension de 9,4. Le pourcentage d'éclosion a été de 67 ± 32 et le poids vif à 70 jours de 313 g en moyenne, avec une fourchette de 142 à 492 grammes. A cette époque, 65 p. 100 des poulets couvés étaient morts. L'âge moyen à la première ponte était de 211 jours au moment où les poulettes atteignaient 1 160 q. La période de couvaison a duré de 3 semaines à 4 mois, selon que la poule pondait ou non et selon le temps passé à couver par la poule. Les pondeuses recherchaient leur nourriture très activement sur les déchets ménagers pendant le jour.La quantité moyenne de nourriture de chaque troupe individualisée par propriétaire était de 550 g, exprimée en poids sec. Sa composition approximative était la suivante: 9,4 p. 100 de protéines brutes, 9,2 p.100 d'extrait éthéré et 5,4 p. 100 de cellulose. Plus de 70 p. 100 de la prise alimentaire provenait de déchets ménagers (27 p. 100 de riz cuit, 30 p. 100 de résidu de noix de coco, 8 p. 100 de brisures de riz et 36 p. 100 d'autres restes de cuisine. Le reliquat venait de l'environnement avec 13 p. 100 de pousses d'herbe. 8 p. 100 de petits métazoaires et 7 p. 100 de riz paddy. Les auteurs ont procédé à des analyses approchées de la composition des végétaux, des refus ménagers et de leurs principaux composants. Les taux de Ca et P des rations étaient faibles dans les villages, tout comme les teneurs plasmatiques de ces minéraux. Mais, pour un régime commercial équilibré, le calcium plasmatique était encore plus bas que celui des volailles hybrides du commerce. Des suggestions sont faites pour accroître la productivité du système d'alimentation à partir des déchets ménagers avec ou sans besoins d'intrants complémentaires.

     

番鸭细小病毒与鹅细小病毒PCR鉴别诊断方法的建立

根据番鸭细小病毒（MDPV）和鹅细小病毒（GPV）基因组的非同源序列各设计了1对引物MDPVF1／R1和GPVF1／R1，建立了一种PCR方法，用该PCR方法分别对GPV、MDPV、鸭瘟病毒（DPV）、鸭肝炎病毒（DHV）、鸭呼肠孤病毒（DRV）、犬细小病毒（CPV）和猫泛白细胞减少症病毒（FPLV）的病毒培养物及其核酸进行扩增。结果，引物MDPVF1／R1仅特异性扩增出MDPV的900bp核酸片段，引物GPVF1／R1仅特异性扩增出GPV的465bp核酸片段。表明，建立的PCR方法可用于GPV和MDPV的鉴别诊断。     

Evaluation of a PCR assay for identification and differentiation of Campylobacter fetus subspecies

Objective To evaluate a polymerase chain reaction assay for identification of Campylobacter fetus and differentiation of the defined subspecies.
Design Characterisation of bacterial strains by traditional phenotyping, polymerase chain reaction, a probabilistic identification scheme and macrorestriction profiling using pulsed field gel electrophoresis.
Procedure The results of identification of 99 bacterial strains as determined by conventional phenotyping or by poly-merase chain reaction were compared. Two of these were type strains of C fetus subsp fetus and C fetus subsp venerealis ; the remaining strains were field isolates putatively identified as C fetus . In cases where the subspecies identity was disputed, isolates were identified by means of a probabilistic identification scheme and by macrorestriction profiling.
Results The agreement between strain identities initially suggested by traditional phenotypic methods and the PCR assay was found to be 80.8%. The polymerase chain reaction proved to be a reliable technique for the species and subspecies identification of C fetus ; equivocal results were obtained in only two instances. Initial misidentifications by conventional phenotyping methods were attributed to methodological differences used in various laboratories.
Conclusion Our results indicate that misidentification of C fetus i n routine diagnostic laboratories may be relatively common. The PCR assay evaluated gave rapid and reproducible results and is thus a valuable adjunctive method for the identification of C fetus and subsequent subspecies differentiation.     

猪传染性胸膜肺炎PCR诊断方法的建立

根据已发表的猪胸膜肺炎放线杆菌APXIV毒素的基因序列，自行设计和合成了二对可扩增448bp和365bp目的片段的引物，成功的建立了检测APP的套式PCR方法。通过对猪肺疫巴氏杆菌、猪链球菌、大肠杆菌、猪嗜血杆菌、猪肺炎支原体和猪丹毒杆菌的DNA进行了PCR检测，结果均为阴性；对猪胸膜肺炎放线杆菌的1、2、5、6、7、9国际标准血清型均扩增出448bp和365bp的特异性条带；检测的敏感度一步PCR可达到5OO个细菌，最低检出DNA浓度可达到0．585ng／mL；套式PCR可达到50个细菌，最低检出DNA浓度可达到58．5pg／mL。另外，对5株从病猪体内分离的猪胸膜肺炎放线杆菌进行了检测，5株均成阳性反应；对10只屠宰猪的肺脏分离物进行了检测，结果1份为阳性。结果表明此法特异性和敏感性均很高，可做为猪传染性胸膜肺炎的快速诊断和流行病学调查的手段。     

羊痘病毒多重PCR检测方法的建立

根据羊痘病毒（CaPV）末端反向重复序列中的一段序列和P32基因序列,设计合成了2对引物,通过确定最佳的PCR反应条件,建立了检测羊痘病毒核酸的多重PCR方法。结果显示,用这2对引物均能扩增出羊痘病毒相应的特异性片段,而用此PCR方法检测口蹄疫病毒和羊口疮病毒,结果均为阴性。与中和试验比较,建立的PCR方法具有更好的敏感性。表明,该PCR方法可对组织病料中的CaPV进行快速检测。     

牛巴贝斯虫实时荧光PCR检测方法的建立

为建立牛巴贝斯虫(B.bovis)的TaqMan实时荧光PCR检测方法,本研究根据GenBank中B.bovis的18S rRNA基因保守序列,设计引物和TaqMan探针,通过优化反应体系,建立检测B.bovis的实时荧光PCR方法.试验结果表明:实时荧光PCR对靶基因的最低检测值为1.31×101 copies/μL,比常规PCR的敏感性高1 000倍;而且与牛的其他血液原虫无交叉反应;组内及组间重复性试验的变异系数均小于3％,具有良好的重复性;在23份被检样品中,实时荧光PCR和常规PCR的检出率分别为52.17％和30.43％.该检测方法的建立为B.bovis的检测提供了一种快速、敏感、特异的技术手段.     

Seroepidemiology of rinderpest in bovids in Sri Lanka using the enzyme linked immunosorbent assay (ELISA) technique

Approximately 0.2% (n=4397) of the bovids (cattle and buffalo) in Sri Lanka were sampled, from June 1992 using a multi-stage sampling procedure. Serum antibodies for the rinderpest virus were detected using the competitive enzyme-linked immunosorbent assay. The age, the agroclimatic zone, the management system practiced in the farms, and the vaccination history of the sampled bovids were studied as potential risk factors for being seropositive.

The prevalence of rinderpest antibodies in non-vaccinated bovids was 3.5% (n=4101). The prevalence was higher in the dry zone (9%; where the outbreak emerged in 1987), compared to bovids in the other zones (1%). Seropositive bovids over three years of age were approximately at fourfold higher chances of being seropositive compared to those that were ≤3 years old. The higher prevalence in older animals is probably due to exposure to the virus during the 1987 epidemic. Bovids from the dry zone (annual rainfall 20 to 35 inches) were at higher odds of being seropositive even after controlling for the possible effects of age, agroclimatic zone, management system and vaccination. The fact that 62% of bovids from the dry zone in this study were reared under extensive management system (free grazing) which allow unrestricted contact between animals, may be the reason for the above finding. A relatively poor response to vaccination observed in vaccinated bovids (seroprevalence=12%; n=296) could be attributed to difficulties in maintaining the vaccine at recommended temperatures in the field. This is the first island-wide study on seroprevalence of rinderpest in Sri Lanka.     

Incidence of brucellosis in Sri Lanka: an overview

Infection by Brucella abortus seems to be a major cause of abortions among cattle and buffaloes in Sri Lanka. The incidence of this disease is more prominent among the animals in the Dry zone of the country raised under extensive management systems. The present low incidence of this disease and the small size of the country may facilitate launching of an effective disease control scheme. The milk ring test (MRT) has proven to be usable in testing milk for the infection at farm level. An ELISA technique could be employed to test the seroprevalence of infection among MRT-positive animals. A program to purchase the diseased animals by the state for slaughter, and a countrywide vaccination program with B. abortus strain RB51 would enable the country’s livestock industry to eventually eradicate this disease.     

Risk factors for bovine mastitis in the Central Province of Sri Lanka

A study of the risk factors associated with mastitis in Sri Lankan dairy cattle was conducted to inform risk reduction activities to improve the quality and quantity of milk production and dairy farmer income. A cross-sectional survey of randomly selected dairy farms was undertaken to investigate 12 cow and 39 herd level and management risk factors in the Central Province. The farm level prevalence of mastitis (clinical and subclinical) was 48 %, similar to what has been found elsewhere in South and Southeast Asia. Five cow level variables, three herd level variables, and eight management variables remained significant (p?相似文献   

鹅圆环病毒实时荧光定量PCR检测方法的建立

为建立鹅圆环病毒(GoCV)快速检测方法,本研究根据GenBank中登录的GoCV全长基因组序列,在其保守基因区设计并合成1对引物,采用荧光嵌合法(SYBR GreenⅠ)建立检测GoCV的实时荧光定量PCR(real-time PCR)方法。以本实验室构建的含有GoCV永康株(GoCV-yk01)全长基因组的重组质粒为标准模板,经退火温度、循环次数等反应条件的优化,绘制GoCV real-time PCR的标准曲线,并进行融解曲线分析。试验结果表明:建立的GoCV real-time PCR方法特异性强,检测范围广(Ct值范围12～32),最低检出病毒拷贝数为1.46×102。该方法能够对组织中病毒进行定量检测,为快速检测GoCV提供了有效的技术手段。     

牛支原体套式PCR检测方法的建立

为建立牛支原体检测方法,本研究采用牛支原体的oppD/F基因的两对特异性引物,建立了牛支原体的套式PCR检测方法.该套式PCR可以从100 ccu/mL(color change unit颜色变化单位)的牛支原体培养物中检出目的片段;同时还可以从病牛肺脏、病牛鼻拭子中扩增出目的片段,而且结果与病原分离结果一致.特异性试验结果表明,该方法与其它支原体无交叉反应,是一种特异性强、敏感性高的牛支原体检测方法.