Comparison of serological tests for detection of Mycoplasma gallisepticum antibodies in eggs and chicks hatched from experimentally infected hens

Specific pathogen free hens and males were experimentally infected with Mycoplasma gallisepticum. Eggs were then collected, and a part was incubated and set for hatching. Mycoplasma cultures were performed on infected adults and antibodies to MG were analysed by use of slide agglutination (SA) test and commercial ELISA tests on adults and chicks sera and on yolks from non incubated eggs. Both ELISA tests could detect antibodies in yolks from non incubated eggs laid three weeks after infection. SA and the three ELISA tests revealed positive sera in chicks hatched from eggs laid as soon as one week after infection.     

Diagnostic utility of egg yolk for the detection of avian metapneumovirus antibodies in laying hens

Surveillance and diagnosis of avian metapneumovirus (AMPV) infection typically involve measurement of serum antibodies. In the current study, eggs instead of serum samples were used for the detection of AMPV antibodies in egg-laying chicken hens by enzyme-linked immunosorbent assay (ELISA). AMPV-free commercial layer hens were experimentally challenged with AMPV strain SC1509 through intravenous or oculonasal administration. Antibody levels were determined by ELISA. AMPV antibodies were detected in egg yolks from challenged hens by 7 days postinoculation (dpi), with the peak titer at 16 dpi. Antibody levels in eggs laid at 28 dpi correlated well (r = 0.93) with sera taken 28 dpi from the same hens. In a field trial of the yolk ELISA, six broiler breeder farms were surveyed, and all tested positive for AMPV antibodies in hen eggs, although positivity varied from farm to farm. Abnormal discolored eggs collected from outbreak farms had significantly higher titers of AMPV yolk antibodies than normal eggs from the same farm, unlike clinically healthy farms, where normal and abnormal eggs had similar antibody titers. These results indicate that diagnosis of AMPV infection by yolk ELISA to detect anti-AMPV antibodies may be a suitable alternative to serologic testing.     

Evaluation of enzyme-linked immunosorbent assay (ELISA) and immunofluorescent antibody (IFA) test for the detection of porcine reproductive and respiratory syndrome virus (PRRSV) antibody in pigs from conventional farms

To evaluate the immunofluorescent antibody (IFA) test and enzyme-linked immunosorbent assay (ELISA) for detecting the porcine reproductive and respiratory syndrome virus (PRRSV) antibody, conventional pigs in PRRSV-positive and -negative commercial farms were examined. Antibody development patterns in ELISA and IFA tests were compared in 3 week old piglets experimentally infected with the PRRSV. The virus was detected from 2 days post infection (PI) and then the antibody titers and S/P ratios rose by both methods. A total of 208 serum samples were collected from 4 PRRSV-negative farms and 210 samples from PRRSV-positive farms, and were tested for the PRRSV antibody by IFA and ELISA. The titer of 64 should be set as the cut-off point in IFA for field sera. Similarly, the cut-off S/P ratio should be set at 0.4 in ELISA. A high degree of correlation was observed between antibody titers by the two methods in these 418 samples, with a correlation coefficient of 0.84. The coincidence rate between the two tests was 84.7% (354/418). In non-coincident cases, ELISA was able to detect the antibody with a low titer in the serum samples which were negative in IFA but from PRRSV positive farms. ELISA was more sensitive than IFA to detect PRRSV infected animals or farms.     

Genetic differences of Ascaridia galli egg output in laying hens following a single dose infection.

Groups of 20-week-old white (Lohmann LSL) and brown (Lohmann Brown) hens were reared under helminth-free conditions and both divided into two subgroups. One subgroup was artificially infected with 250 embryonated Ascaridia galli eggs, the other subgroup were kept as uninfected controls. During the following 12-month laying period individual faecal Ascaridia egg counts (FEC) were performed and bodyweight measured at monthly intervals. Laying performance and egg weights were determined daily. The mean FEC were significantly (P < 0.01) higher in infected white hens than in infected brown hens. The growth rate of the white hens was significantly (P < 0.05) higher in the control group, whereas in brown hens no significant group difference was observed. The laying performances and egg weights did not differ significantly (P > 0.05) between control and infected animals for either line. The estimated repeatabilities for mean log(10) FEC of different samples were reasonably high (0.16-0.54). Heritabilities for mean log(10) FEC were between 0.13 and 0.19 for white hens and between 0.0 and 0.10 for brown hens. Thus, it should be possible to select for A. galli resistance in chickens, which will be of importance for birds kept in alternative and organic farming systems.     

Salmonella enteritidis and other Salmonella in laying hens and eggs from flocks with Salmonella in their environment.

Seven Canadian layer flocks with Salmonella enteritidis in their environment were investigated to determine the numbers of hens infected with S. enteritidis, the localization of S. enteritidis in organs of infected hens and the numbers of S. enteritidis-infected eggs produced by two affected flocks. By a microagglutination test (MAT) using S. pullorum antigens, these flocks had more seropositive hens (mean 51.9 +/- 16.9%) than two Salmonella-free flocks (mean 13.0 +/- 4.2%). Culture of tissues of 580 hens (433 seropositive) from the seven flocks detected 26 (4.5%) S. enteritidis-infected hens from two flocks. In one flock, 2/150 hens were infected with S. enteritidis phage type (PT) 8, which was confined to the ceca, and no Salmonella spp. were isolated from 2520 eggs (one day's lay). In the second flock, where 24/150 hens were infected with S. enteritidis PT13, extraintestinal infection was found in nine hens and involved the ovaries and/or oviduct in two hens. Salmonella enteritidis PT13 was isolated from one sample of egg contents and from one sample of cracked shells from among 14,040 eggs (one day's lay) from this flock. The overall prevalence of S. enteritidis-contaminated eggs from the two flocks with infected hens was less than 0.06%. Other Salmonella spp. isolated were S. heidelberg from 58 hens (10%), and S. hadar, S. mbandaka and S. typhimurium from one hen (0.2%) each. The MAT with antigens of S. pullorum had a sensitivity of 81% and a specificity of 24% for detecting S. enteritidis-infected hens.(ABSTRACT TRUNCATED AT 250 WORDS)     

Vertical transmission of reticuloendotheliosis virus in breeder turkeys

Epizootiological studies were conducted on a commercial turkey breeder flock naturally infected with nondefective reticuloendotheliosis (RE) virus. Although RE virus was isolated from 27 (46%) of the 59 hens studied, only 4 of the 45 hens tested transmitted RE virus to progeny during a 6-week observation period and the overall transmission rate was 1.8%. The transmitter hens were of two types: three hens were consistently viremic and antigenemic and lacked antibody; one hen was viremic but lacked detectable viral antigen and possessed antibody. Toms appeared to play no role in vertical transmission of the infection. Of several tests evaluated for detection of transmitter hens, the direct enzyme-linked immunosorbent assay on albumen was probably best, since it detected three of four transmitter hens, detected relatively few nontransmitter hens, and had the best consistency of any test. No significant differences in hatchability were found between eggs from viremic and non-viremic hens. These findings can be utilized in the development of programs for eradication of RE virus from turkey breeding flocks.     

Detection of antibodies in serum and egg yolk following infection of chickens with an H6N2 avian influenza virus.

Active serologic surveillance programs to detect avian influenza viruses (AIVs) in table egg-laying chickens have been initiated by several states as a response to the economic threat posed by these viruses. Most outbreaks of avian influenza in domestic poultry are caused by mildly pathogenic AIVs. In the study reported here, infection by an H6N2 AIV was used as a model of mildly pathogenic AIV infections in egg-type chickens. The total number of eggs laid by 5 control hens was 619 or 0.904 eggs/day/hen, whereas the total number laid by 10 infected hens was 1,018 or 0.743 eggs/day/hen. The difference in egg production between the 2 groups was not statistically significant (P = 0.38). Anti-influenza antibodies were monitored by use of an agar gel immunodiffusion test and an ELISA for a period of 20 weeks after inoculation. Antibodies in serum developed sooner, peaked at higher levels, and remained at higher levels than did antibodies found in egg yolk, as indicated by ELISA results. For infected chickens, the correlation between serum and egg yolk ratios was 0.66. Serum samples would appear to be preferable to egg yolk samples for surveillance programs intended to identify chicken flocks that may have been infected by an AIV weeks or months before samples are collected.     

Detection of Salmonella in spent hens and eggs associated with foodborne infections

About 16,000 spent hens from 23 farms in the northern area of Japan were purchased in 1996, 1997, 1998, and 1999 to isolate Salmonella in two poultry processing plants. Salmonella was detected in 12 of 23 farms (52.2%). In particular, the serotypes Enteritidis and Infantis were detected in four and three farms, respectively. The prevalence rates in the hens' ceca, immature eggs, and the yolk of mature eggs in oviducts were 14%, 7.2%, and 6.8%, respectively. A total of 23 serotypes were detected. The major serotypes of the strains were Enteritidis, Corvallis, Typhimurium, and Infantis, but most of the strains were untypable. In the same area during 1992 to 1996, Salmonella was detected in eggs associated with four outbreaks of Salmonella Enteritidis infection and one outbreak of Salmonella Infantis infection. The ratio of contamination was approximately 1%, and the level was estimated to be 93 MPN(most probable number)/100 g in one outbreak. In farms that produced the eggs associated with all of the five outbreaks of Salmonella, the serotype Enteritidis or Infantis was isolated from hens. Farms where Salmonella was not detected were not related to any of the outbreaks.     

Detection of antibody to Salmonella enteritidis and S typhimurium in the yolk of hens' eggs

Enzyme-linked immunosorbent assays (ELISAs) have been developed to detect IgG antibodies to Salmonella enteritidis and S typhimurium in the yolk of hens' eggs. Better discrimination and more consistent results were obtained between eggs from experimentally infected and uninfected hens by using saline-dilution of yolk rather than chloroform extraction. Threshold absorbance values were determined in three salmonella-free flocks, and on the basis of these results ELISA optical density values greater than 0.25 were considered to be positive for antibodies to salmonella. Four flocks with a history of salmonella infection were examined; three contained birds which were seropositive for S enteritidis by ELISA and from which S enteritidis was isolated, and a large proportion of eggs from these birds contained antibody to S enteritidis. Eggs from the fourth flock had no detectable antibody, although serum antibody was detected in some birds. No salmonellae were isolated from the yolks of the eggs from any of the four flocks.     

Infection dynamics of Ascaridia galli in non-caged laying hens

The infection dynamics of Ascaridia galli in laying hens was investigated in six commercial non-caged flocks. Three flocks were managed in accordance with the regulations for organic production and had outdoor access, whereas three flocks were housed indoors in aviaries or traditional floor systems. Faecal egg counts and total worm burdens were determined at specified intervals during the first 50 weeks of the production period. In two conventional flocks the efficacy of flubendazole on lumenal stages was investigated. All flocks became infected following the arrival of the birds (post placement) with residual infective eggs derived from the previous flock. In four flocks (two organic and two conventional) parasite eggs were first detected in faeces 6-7 weeks post placement, whereas parasite eggs were not detected until after 17-18 weeks in two flocks. This delay was observed in two of three flocks that were housed in barns that had been thoroughly cleaned and disinfected by chlorocresol. In three flocks (two conventional and one organic) flubendazole was administered to the birds in the drinking water for approximately one week. Both conventional flocks were dewormed twice approximately 20 weeks apart, whereas the organic flock was dewormed only once about 40 weeks post placement. Parasite eggs reappeared after deworming in all flocks, often within 2-4 weeks, followed by a rapid increase in parasite egg expulsion. Our results suggested impairment of host immunity post treatment, as the egg counts exceeded pre-treatment levels after 7-8 weeks on both conventional farms. Accordingly, the way by which anthelmintics and/or disinfectants are used in non-caged chicken flocks must be refined.     

Diagnosis of bovine brucellosis by enzyme immunoassay of milk

Enzyme-immunoassays using lipopolysaccharide as antigen were developed for the detection of bovine IgG1, IgG2 or IgA Brucella antibodies (Ab) in milk. The results of these tests were compared with those of the milk ring test (MRT) by analyzing 3212 bulk milk samples from farms located in regions where brucellosis is prevalent. Among the 105 herds detected by ELISA and/or MRT, 29 infected herds were detected by ELISA only. The 40 MRT-positive herds were also ELISA positive. Five herds became infected during the study and were detected by ELISA 15 days to 6 months prior to detection by MRT. The ELISA IgG1 titration (IgG1 ELISA) detected 92.8% of the herds found positive in the three ELISA assays. The concomitant use of IgA ELISA raised the sensitivity to 100% but slightly decreased the specificity. The IgG2 ELISA did not improve the diagnosis. The sensitivity of MRT and IgG1 ELISA was compared by testing successive dilutions in negative milk of 110 individual MRT positive milks samples. On average, IgG1 ELISA was 22 times more sensitive than MRT.     

猪圆环病毒2型高免卵黄抗体的研制与应用

为制备猪圆环病毒2型(porcine circovirus serotype2,PCV-2)高免卵黄抗体,并对其治疗效果进行研究,通过自制猪圆环病毒2型(PCV-2)灭活苗,免疫SPF产蛋鸡,收集高免蛋,制备PCV-2高免卵黄抗体,对高免卵黄抗体进行无菌检验、活体动物安全性检测,用琼脂扩散方法进行效价检验和人工感染保育猪治疗试验,并将其应用于临床实践,观察其治疗效果。结果抗体效价达到1∶64;人工感染治疗试验治愈率达100%;临床试验治愈率为96%,应用效果良好。     

Neutralizing antibodies to CELO and avian adenovirus-associated viruses in the albumen of chicken eggs

Neutralizing antibodies to CELO virus and to avian adenovirus-associated virus (A-AV) were detected in the albumen of eggs from four hens inoculated with these viruses. The antibody concentrations of serum, yolk, and albumen were determined before inoculation and at various times postinoculation (PI) by enzyme-linked immunosorbent assay (ELISA) and virus-neutralization (VN) tests. The antibody concentration in albumen was 0.3% to 1.0% of that detected in serum and yolk. Uninoculated hens showed no detectable antibody in serum, yolk, or albumen. It is suggested that the presence of antibody in the egg albumen may play a role in egg-transmission of viruses.     

抗猪流行性腹泻病毒卵黄抗体的制备及应用

本试验利用猪流行性腹泻病毒(porcine epidemic diarrhea virus,PEDV)SC株免疫产蛋鸡,收集高免卵黄,采用水稀释法和水稀释-盐析法获得鸡源抗PEDV卵黄抗体,采用已建立的ELISA方法对卵黄抗体效价进行测定。以3日龄无母源抗体的易感仔猪为试验动物,对抗PEDV卵黄抗体的治疗效果及安全性进行测定,并进一步利用自然发病猪场的治疗效果进行验证。结果表明,经PEDV SC株细胞毒免疫的鸡可在2次加强免疫后15 d产生高水平的特异性抗体。在实验室治疗试验中,攻毒治疗组仔猪的存活率达60%,攻毒对照组存活率为20%;用于自然发病猪场时,饲喂抗PEDV卵黄抗体饲料的仔猪存活率亦为60%,而自然发病对照组的存活率为10%。以上结果表明抗PEDV卵黄抗体对感染PEDV的仔猪有一定治疗作用。     

A comparison of coprological, serological and molecular methods for the diagnosis of horse infection with Anoplocephala perfoliata (Cestoda, Cyclophyllidea)

Anoplocephala perfoliata (Cestoda, Cyclophyllidea), the commonest intestinal tapeworm of horses, can cause colic, intussusceptions, ileal impactions and intestinal perforations. Common diagnostic techniques for A. perfoliata infection, i.e. coprology and serology, show inherent limitations in terms of sensitivity and specificity and new approaches are thus required. Hence, the present study compared the reliability of coprological, serological (i.e. ELISA) and molecular (i.e. nested PCR) methods in detecting A. perfoliata infection in naturally infected horses and in horses treated with a combination of ivermectin and praziquantel. Of 42 horses subjected to coprological examination, 16 and 26 resulted negative and positive, respectively for the presence of A. perfoliata eggs at the coprological examination. The 26 coprologically positive animals were also positive by nested PCR. Fifteen out of the 16 horses coprologically negative were negative at the molecular assay, while one yielded a PCR product detectable on an agarose gel. Eighteen out of 26 positive horses were treated with a combination of ivermectin 18.7 mg/g and praziquantel 140.3mg/g and resulted subsequently negative by coprology and nested PCR performed 2 weeks after treatment. All infected and untreated animals had a high ELISA test optical density indicating high infection intensity and associated risk of colic. However, high optical density values were also obtained in four horses post-treatment and in three horses that were negative on molecular and coprological analysis. The results of the present work indicate that the nested PCR assay represents a valid method for the specific molecular detection of A. perfoliata in faecal samples collected from naturally infected horses and may have advantages over coprological and serological approaches for diagnosing A. perfoliata infection.     

Campylobacter jejuni contamination of eggs

Contamination of commercial table eggs with a fecal suspension containing 4.4 X 10(6) CFU/g Campylobacter jejuni resulted in shell penetration in 3/70 eggs and recovery of the organism from homogenized egg contents in 1/70 eggs. Viability of C. jejuni on the shell surface was retained for only 16 hours, attributed to desiccation of the fecal suspension. A field survey of three commercial laying farms and their associated egg-packing plants showed that hens demonstrated to be fecal shedders of C. jejuni (12% to 62% incidence) did not produce infected eggs. The organism could not be detected in the environment of the packing plant, including grading machinery and effluent.     

The relationship between the magnitude of the specific antibody response to experimental salmonella enteritidis infection in laying hens and their production of contaminated eggs.

Detecting infected laying flocks is a vital part of many efforts to control egg-associated transmission of Salmonella enteritidis to humans. The relationship between the development of a specific antibody response in infected hens and the deposition of S. enteritidis in eggs is important for establishing the epidemiologic relevance of serologic testing methods. In two trials, laying hens were infected with large oral doses of phage types 13a and 14b isolates of S. enteritidis. Approximately 38% of all infected hens produced at least one contaminated egg, at an overall incidence of 5.2%, between 3 and 23 days postinoculation. As determined by enzyme-linked immunosorbent assay with an S. enteritidis flagellar antigen, 91.7% of inoculated hens produced specific serum antibodies. Although hens with very high antibody titers were associated with a significantly elevated frequency of egg contamination, a consistently direct relationship was not evident between the magnitude of the antibody responses of individual hens and the frequency at which they laid contaminated eggs. Accordingly, although serologic tests can be valuable screening tools for preliminary detection of S. enteritidis infections in poultry, the magnitude of the antibody responses detected in individual hens may not predict the overall risk of egg contamination associated with particular laying flocks.     

安徽省部分地区鸡群禽偏肺病毒感染的血清学调查

为掌握禽偏肺病毒(aMPV)在安徽省鸡群中的感染状况,采用酶联免疫吸附试验(ELISA)对安徽省合肥、亳州、定远、舒城等地区的9个鸡场、7个不同品种(系)鸡群的296份血液样本进行了aMPV血清抗体检测。结果表明,所有被检鸡场均有aMPV感染,鸡场阳性率最高达100%,最低为20%;各品种(系)鸡均有感染,感染率最高的是青脚麻肉鸡,其次分别为科宝肉鸡、海兰蛋鸡、禽粤黄蛋鸡、淮南麻黄鸡、黄羽土鸡和新广麻肉鸡;其中蛋用型鸡血清样本总体阳性率为88.7%,明显高于肉用和兼用型鸡;公鸡和母鸡血清抗体阳性率均较高。研究结果表明,安徽省鸡群aMPV的感染已广泛存在,且不同地区、品种(系)、用途和性别的鸡群均较严重,应根据感染状况尽早制定相应的防控对策。     

Use of an enzyme-linked immunosorbent assay to measure antibody levels in turkey breeder hens, eggs, and progeny following natural infection or immunization with a commercial Bordetella avium bacterin

Twelve large white turkey hens were immunized with a commercially available Bordetella avium bacterin. Hens and eggs were tested using an enzyme-linked immunosorbent assay (ELISA) to determine the response to the bacterin. Three hundred poults were then obtained from two commercial flocks, the hens of one flock having been immunized with the same bacterin used on the group of 12 turkeys. Titers of the poults were monitored for 7 weeks, and poults were challenged by exposure to infected poults at 1, 7, 14, and 21 days post-hatch. Hens produced an antibody response following immunization, with a parallel antibody response being detected in eggs. Maternal antibodies were present in poults from immunized hens. Poult titers declined to near the level of poults from unimmunized hens by 14 days of age. Poults from immunized hens challenged at 1 and 7 days were resistant to development of clinical disease and gross lesions, whereas all poults from unimmunized hens exhibited clinical signs and gross lesions. After 14 days, the resistance of both groups to development of clinical disease, became near equal, neither group being affected as severely as the unimmunized hens challenged at days 1 and 7. Six commercial turkey breeding flocks and their progeny that had not been vaccinated for B. avium and had no history of B. avium infection were evaluated with the B. avium ELISA. There were variations between the flocks, with poult titers reflecting those found in the hens.