Use of an alkaline phosphatase-labelled probe for the detection of Mycoplasma synoviae in chickens.

Short nucleotides directly labelled to alkaline phosphatase (SNAP probes) are an interesting alternative to digoxigenin-labelled probes (DIG probes), because they reduce the number of steps necessary in dot blots for the detection of DNA or amplificate. This study examined the questions whether a SNAP probe might not only save time, but also increase the sensitivity of another PCR-based DNA probe test using a digoxigenin probe. Amplificates obtained by multispecies polymerase chain reaction (PCR), with either purified genomic DNA or DNA extracted from tracheal swabs taken in chicken flocks, were detected by both methods. The results for the clinical specimens were compared to culture. Under stringent conditions, the specificity and sensitivity obtained with the SNAP probe were comparable to the results obtained with the DIG probe. The quantities 10 fg (SNAP probe) and 100 fg (DIG probe) of purified Mycoplasma synoviae DNA were detected after amplification, but more positive clinical specimens were detected with the DIG probe. Under non-stringent conditions sensitivity with purified DNA did no change, but the coloration of the dots improved markedly, and more positive specimens could be detected with the SNAP probe than with the DIG probe, truly positives as confirmed by culture. Because cross-reaction with Mycoplasma gallisepticum and Mycoplasma imitans, two species with DNA that was also recognized by the multispecies primers, occurred under non-stringent conditions, it was concluded that, to take the full advantage of SNAP probes, their use in combination with species-specific primer pairs is recommended. PCR as a method for mycoplasma detection is however, always accompanied with serological and cultural methods. When a M. synoviae mono-infection is likely by serological results, non-stringent dot blot conditions and use of the SNAP probe will ease and improve the detection of mycoplasma.

Experiences with multispecies polymerase chain reaction and specific oligonucleotide probes for the detection of Mycoplasma gallisepticum and Mycoplasma synoviae.

Amplified fragments of the rDNA coding for 16S rRNA of Mycoplasma gallisepticum (MG) and Mycoplasma synoviae (MS) were blotted on nylon membranes, followed by dot-blot detection with two species-specific digoxigenin-(DIG)-labeled oligonucleotide probes. The sensitivity and specifity of the tests were determined in titration studies with purified homologous and heterologous DNA. With the detection protocol used, the MSYV8/31 probe showed 100% specifity for MS, while both MG and the related species Mycoplasma imitans were recognized by the MGAV8/31 probe. Both DIG-labeled oligonucleotides gave positive results in the colorimetric assay with 10 to 100 ng homologous non-amplified DNA and polymerase chain reaction (PCR) amplificates of 100 fg homologous template DNA. There was no reaction with heterologous strains when amplificates starting with a 106-fold amount of template DNA (100 ng) were tested in dot-blots. The suitability for field samples was demonstrated with tracheal swabs from turkeys and chickens, and the results were compared with mycoplasma growth in cultures of the same swabs. Both tests had an accuracy of over 95%, a high sensitivity and specificity, and high predictive values of positive or negative results. There was no significant difference between the results obtained by the two methods. PCR in combination with dot-blotting is a relatively simple method for the detection of mycoplasma infections, and a valuable extension of current diagnostic tools.

A comparison of a commercial PCR-based test to culture methods for detection of Mycoplasma gallisepticum and Mycoplasma synoviae in concurrently infected chickens.

The suitability of commercial PCR-based test kits for the detection of either Mycoplasma gallisepticum (MG) or M. synoviae (MS) was compared to detection by culture. The MG and MS kit detected six and five homologous strains respectively in broth cultures and there were no reactions with thirteen het-erologous species including M. imitans, a species phylogenetically closely related to MG. Tracheal and lung/air-sac swabs were collected from twenty 17-week-old commercial pullets which were seropositive for MS and were compared for detection of MS by kit and by culture. The results were in agreement for 13 positive and 22 negative swabs, while the remaining five swabs were either PCR-positive only (two) or culture-positive only (three). Tracheal swabs taken from seventy-six 31-week-old layers from a MS seropositive commercial flock which had been experimentally infected with MG were used to compare the MG DNA probe kit and culture. Of 76 swabs 39 were MG positive and 12 were negative by both methods. MG was detected by PCR test only in 23 other specimens, while only two swabs were negative by PCR but positive by culture. The difference between the detection methods was significant (McNemar test, P < 0.001). Concurrent MS infection was detected by the PCR-based kit in 45 of these hens.

[Evaluation of different broiler housing conditions on the basis of clinical, pathological, anatomical and laboratory diagnostic criteria--a summary]. / Bewertung verschiedener Haltungsbedingungen von Broilern anhand klinischer, pathologisch-anatomischer und labordiagnosticher Kriterien-Eine zusammenfassende Darstellung.

In a cooperative study, the impact of different broiler housing conditions (see communication BUCHENAUER) on clinical, post-mortem and laboratory diagnostic (microbiological, parasitological, serological, immunological) parameters was investigated in the course of three different fattening period. Clinically, it was observed, that lower stocking densities allowed a relatively higher motility of the birds. On the other hand, necropsy findings, microbiological, parasitological, serological and immunological findings gave no indications that these parameters were influenced statistically significant by either one of the broiler keeping systems.

Oxygen concentration and asexual development of Eimeria tenella in cell cultures.

Primary chicken kidney cells in Flexiperm cultures were either inoculated with Eimeria tenella sporozoites or incubated as noninoculated controls. Oxygen concentration was reduced (10 or 15 vol% O2, 5 vol% CO2) or increased (25 or 30 vol% O2, 5 vol% CO2) in a triple gas incubator (Heraeus B 5061 EK/O2) and retained in a CO2-air incubator (20 vol% O2, 5 vol% CO2) 24 hours post inoculation (hpi). Mature second generation schizonts (mS2) were counted microscopically at 120 hpi and numbers were compared either as mS2 or mS2/cm2 confluent cells. Asexual development of Eimeria tenella was neither stimulated nor inhibited by different oxygen concentrations, indicating that higher numbers of schizonts in cultures under reducing conditions reported earlier are probably a result of increased invasion rates of sporozoites.

Clinical selection criteria for secondary examinations of the health-status of broiler flocks.

Results of post-mortem and laboratory examinations of broilers sampled randomly or selected by clinical symptoms were compared. The male:female-ratio was in all cases nearly equal for randomly sampled and selected birds. Regarding post-mortem findings, differences dependent on selection criteria were observed: in randomly-sampled birds the frequency of breast-blisters and plantar inflammations was higher than in selected broilers, whereas in the latter osteodystrophy and bursal atrophy were seen more frequently. Alterations of joints and tendon-sheaths caused by a reovirus-arthritis in one flock were seen in same frequency in randomly sampled and selected birds, respectively. Parasitological examinations revealed the highest coccidial lesion scores in selected birds with lameness or slightly retarded growth but when dwarfism birds were included into calculations, there was no significant difference among both groups. Results of bacteriological and serological examinations showed no differences between the groups.

A study of the life cycle of Eimeria praecox, Johnson 1930.

The life cycle of a strain of Eimeria praecox derived from a single oocyst which was obtained from litter removed from a broiler farm in West-Germany was examined. Four generations of schizonts were observed, maturing at 24 hpi, 40 hpi, 56 hpi and 68 hpi, respectively. Mature gamonts were seen at 80 hpi to 84 hpi, and oocysts were detected in the faeces at less than 84 hours postinfection in two of the five propagations of the strain. Results are compared with previous descriptions of the life cycle of E. praecox.

[Subclinical coccidiosis in broilers and pullets]. / Subklinische Kokzidiosen bei Broilern und Junghennen.

In the period from 1985 to 1987, 24 broiler crops (12 houses; one integration and 3 farms) and 9 pullet flocks (9 houses, 4 farms) were examined for parasites. Intestinal lesion scores and the number of parasites in the intestinal lumen (semiquantitative estimation) were recorded, and the Eimeria species determined when possible (broilers crops: 3rd and 5th week, pullet flocks 4th, 8th, 12th and 18th week). Additionally, the quantity of parasites in litter or faecal samples was examined in regular intervals. Clues for economic damage were only found in broiler crops with increased numbers of coccidial oocysts per gram litter in the 5th week of the fattening period. Eimeria tenella and E. acervulina were the dominating species in broiler chickens and also pullets, E. maxima oocysts however were only casual findings. No ectoparasites, helminths or other protozoa than Eimeria were observed. With regard to the intestinal lesions and the quantities of parasites in the intestine no significant differences were seen when comparing selected broiler chicks and birds taken at random. In pullet flocks the examination of random samples was the superior method, because only freshly dead bodies, collected in insufficient numbers, were suitable for diagnostics. In 3 broiler crops and one flock of replacement pullets kept on a wired floor no Eimeria were diagnosed.

A comparison of two techniques for the quantitative determination of oocysts in faecal samples.

Coccidial oocysts (Eimeria tenella) were counted in faecal samples (n = 2 * 101) from two broiler anticoccidial battery trials. A direct flotation technique using zinc sulphate solution and an indirect technique using sodium chloride solution for the examination of samples stored in potassium dichromate were compared. Both techniques were correlated (P less than 0.001) and showed little difference in sensitivity. With the indirect method, oocyst counts were slightly lower (-1.53%), but the difference between the experimental groups was not significant (Wilcoxon test (pair difference test), p less than 0.083). Advantages and disadvantages of both methods are discussed.