Detection of avian metapneumovirus subtypes in turkeys using RT-PCR.

This study investigated the prevalence of avian metapneumovirus (aMPV) and the detection of molecular subtypes of field strains of the virus using RT-PCR in clinically healthy turkeys and those showing signs of respiratory disease. In the RT-PCR examination of 624 tracheal tissue samples collected from a local turkey abattoir, 2.9 per cent (18/624) of samples tested positive. In the examination of tracheal swab samples collected from flocks with respiratory problems, 18 of 20 samples tested positive. When the results were assessed at flock level, aMPV infection was detected in only one of the 23 clinically healthy turkey flocks, whereas all four flocks with respiratory problems were infected. Molecular typing using primers specific to the attachment glycoprotein (G) gene showed that all 36 positive samples belonged to subtype B. Partial sequence analysis of DNA samples showed 95 per cent homology between the field types and the reference strain aMPV subtype B. Whereas clinically healthy turkeys had been vaccinated with a subtype A virus vaccine, the flocks with respiratory problems had been vaccinated with a subtype B virus vaccine. Despite four blind passages of RT-PCR-positive samples on Vero and chicken embryo fibroblast cells, no cytopathic effect was detected by microscopic examination.

Detection of mycoplasma species in turkeys by culture and polymerase chain reaction.

The purpose of the present study was to investigate the presence of pathogenic mycoplasma species in the turkey population of Turkey. Tracheal samples randomly collected from a total of 624 apparently healthy meat-type turkeys at a commercial abattoir located in the north of the country were examined by culture and genus- and species-specific polymerase chain reaction (PCR) assays for mycoplasma. In the direct plating onto solid specific media, mycoplasma growth was observed from 1.4% (9/624) of the samples, which were confirmed to belong to the Mycoplasma genus by genus-specific PCR. Mycoplasma iowae (MI) and M. meleagridis (MM) were identified by the species-specific PCR from eight and one of the samples, respectively. However, genus-specific PCR amplification was obtained from 2.6% (16/624) of the samples which produced turbidity in the liquid media. Interestingly, these positive samples were different from those obtained from solid agar and mycoplasma growth was not observed when the broth samples were inoculated onto solid media. In the species-specific PCR analysis of the broth samples, MI, MM and M. gallisepticum were identified from twelve, two and two samples, respectively. The inconsistency between the results obtained from liquid and solid media raises questions about the efficiency of isolation procedures for mycoplasma and this warrants further investigation.

Evaluation of immunomagnetic separation-polymerase chain reaction in direct detection of Brucella abortus and Brucella melitensis from cheese samples.

The current study was carried out to assess the use of immunomagnetic separation-polymerase chain reaction (IMS-PCR) in direct detection of Brucella abortus and B. melitensis from soft cheese and to examine a relatively small number of field samples for the presence of these species. Two methodologies, one with IMS and the other without IMS, were employed for recovery of the Brucella species from cheese samples. IMS in conjunction with the PCR assay was determined to detect as low as 3x10(2) bacteria/mL, while the limit of detection with the other extraction procedure was 3x10(3) bacteria/mL. In the analysis of 40 cheese samples collected from various markets, only B. abortus was detected by PCR using both DNA extraction procedures in two (5%) samples. No positive results were obtained by culture and B. melitensis was not found in any cheese samples examined. The results suggest that this technique is promising owing to its pace and high sensitivity and should aid in direct detection of Brucella species from complex food samples.

Prevalence and distribution of tropical theileriosis in eastern Turkey.

This study was carried out to determine the prevalence and distribution of tropical theileriosis in cattle in eastern Turkey by microscopical, serological and molecular methods. A total of 1561 whole blood, 1505 serum and 1483 blood smear samples were collected from cattle of various breeds and ages in 11 towns of Eastern Turkey. Theileria annulata piroplasm DNA extracted from cattle blood was amplified by polymerase chain reaction (PCR) using species-specific primers. Serum antibodies against T. annulata were investigated by indirect fluorescence antibody test (IFAT). Blood smears were examined for Theileria piroplasms by microscopical examination (ME). In the examination of DNA extracted from 1561 blood samples, an amplicon with the size of 721bp was obtained in 37.8% (590/1561) of these samples. Serum antibodies against T. annulata and piroplasm of Theileria spp. were detected in 34.9% (526/1505) and 19.7% (293/1483) of the samples, respectively. The differences between ME and PCR results and between ME and IFAT results were statistically significant (P < 0.05). In contrast, there was no significant difference between the PCR and IFAT results. A total of 179 ticks (136 female; 43 male) belonging to Hyalomma spp. were collected from cattle from three towns. Ticks were identified to be Hyalomma anatolicum anatolicum on the basis of morphological features.

Investigation of arcobacters in meat and faecal samples of clinically healthy cattle in Turkey.

AIMS: To investigate the presence of Arcobacter spp. in minced beef meat (n = 97) and rectal faecal samples (n = 200) collected from cattle immediately after slaughter at a local abattoir in Turkey. METHODS AND RESULTS: Meat samples were examined using three different isolation procedures (CAT-supplemented media, de Boer arcobacter isolation method and membrane filtration method), but only one method (CAT-supplemented media) was employed for faecal samples. The isolated Arcobacter strains were identified by genus- and species-(multiplex) specific PCR assays. Arcobacter spp. were isolated from 5 and 9.5% of meat and faecal samples respectively. Although the only Arcobacter sp. found in meat samples was Arcobacter butzleri, all three pathogenic species--A. butzleri, A. cryaerophilus and A. skirrowii--were detected in the rectal swabs. No Arcobacter was isolated when the de Boer method was used for minced meat samples but the same five meat samples were found positive for arcobacters when CAT-supplemented media and membrane filtration method were used. CONCLUSIONS: The membrane filtration method was found to be superior to the CAT-supplemented media, because it led to a reduction in competing microflora. However, the necessity for one filter and medium for each sample makes this method somewhat expensive. The multiplex-PCR (m-PCR) assay shortened significantly the time required for the identification of Arcobacter spp. and also removed the possibility of false positive results due to other campylobacteria. SIGNIFICANCE AND IMPACT OF THE STUDY: This study reports the isolation of Arcobacter spp. in cattle for the first time in Turkey. The m-PCR assay enables the identification and differentiation of all arcobacters simultaneously in one-step PCR.

Detection of Chlamydophila abortus in ovine milk by immunomagnetic separation-polymerase chain reaction.

The present study was carried out to investigate the presence of Chlamydophila abortus, the causative agent of ovine enzootic abortion, in milk samples collected from sheep flocks with and without the history of abortion in Eastern Turkey by means of immunomagnetic separation (IMS) in conjunction with the polymerase chain reaction (PCR). A total number of 201 milk samples collected from 10 flocks with abortion and four flocks without abortion were tested. In the analysis of the milk samples by IMS-PCR, correct amplification was obtained with three (1.5%) samples originating from one flock with abortion. In the digestion of PCR positive products by AluI, restriction profiles observed in all three samples were determined to be the same as C. abortus S26/3.

Abattoir-based survey of contagious bovine pleuropneumonia in cattle in Turkey.

Blood samples collected from 945 cattle at four local abattoirs in Turkey were examined for contagious bovine pleuropneumonia (CBPP) by the complement fixation test (CFT) and competitive ELISA (cELISA). In addition, the carcases of the animals were examined macroscopically at the abattoirs and 62 lung samples which had lesions suggestive of CBPP were collected for bacteriological culture. To identify suspicious isolates the PCR was used in addition to the routine biochemical tests. By the CFT, two of the 945 serum samples were seropositive, and by the cELISA, four of them were seropositive. In the bacteriological culture of the lungs, growth was observed in 18 (29 per cent) of the samples by the observation of turbidity in the broths. However, when these broths were inoculated into an agar base, growth was observed in only three (4.8 per cent) samples. These isolates were identified as Mycoplasma species on the basis of biochemical tests. In the PCR analysis of DNA extracted from the broths, none of the isolates was identified as Mycoplasma mycoides subspecies mycoides small colony or one of the members of the M mycoides cluster, but amplification was obtained in only eight (44.4 per cent) of 18 samples, using Mycoplasma-genus specific primers. These DNA samples were examined further with primers specific to 16S rRNA and were then sequenced and compared with the databanks; DNA homologies at different levels were observed in five samples, with Mycoplasma alkalescens, Mycoplasma canadense, Mycoplasma bovis and Mycoplasma bovigenitalium.

Seroprevalence of coxiellosis in cattle, sheep and people in the east of Turkey.

Serum samples collected randomly from 416 cattle in 48 herds, and 411 sheep in 47 flocks, in eight different locations in the east of Turkey between June and December 1998, were examined by indirect fluorescent antibody test (IFAT) to determine the prevalence of Q fever. The age, sex, breed, tick control and abortion history of the animals were also recorded. In addition, 102 serum samples were collected from apparently healthy people who were at risk of contracting the disease, such as farmers, veterinarians, abattoir and laboratory workers, and veterinary students. Seropositivity was observed in 5-8 per cent (24/416) of the cattle in 17 (35-4 per cent) of the herds and in 10-5 per cent (43/411) of the sheep in 21 (44-7 per cent) of the flocks. Eight of the 102 people were seropositive, with the highest prevalence (12-0 per cent) in farmers and abattoir workers. All the seropositive farmers had seropositive animals. None of the laboratory workers or veterinary students was seropositive.