Simultaneous detection of Clostridium perfringens and Clostridium colinum by duplex-polymerase chain reaction.

In this study, we provide a protocol for detection of Clostridium colinum and Clostridium perfringens by the single-tube duplex-PCR (dPCR) test for simultaneous and specific detection of both bacteria from pure cultures and fecal samples spiked with these pathogens. Specific primers for each pathogen were selected that amplified products of predicted sizes from bacteria in the dPCR as well as in the single-tube PCR (sPCR) assays. The sensitivity and specificity of dPCR assay were compared with those of the sPCR. No product amplification was obtained with DNA from reference strains belonging to the genus Clostridium (except C. colinum and C. perfringens) and isolates belonging to other genera using these primer sets. The dPCR assay was as sensitive as the sPCR assay because bacterial detection limits were similar in both assays. The detection limits of sPCR and dPCR in bacterial suspension were 20 and 25 cfu/mL for C. colinum and C. perfringens, respectively. Meanwhile, in the presence of feces the sensitivity of both assays decreased to a detection limit of 1.25 × 10(4) and 1.94 × 10(4) cfu/g of feces for C. colinum and C. perfringens, respectively. In summary, dPCR assay holds potential to be an economical and rapid diagnostic method for the simultaneous detection of C. colinum and C. perfringens in pure cultures and could be used to screen fecal samples for the presence of these pathogens.

Simultaneous detection of astrovirus, rotavirus, reovirus and adenovirus type I in broiler chicken flocks.

Enteric diseases cause substantial economic losses to the poultry industry. Astroviruses, rotaviruses, reoviruses, and adenovirus type 1 have been reported as a significant cause of intestinal symptoms in poultry. In the present study, intestinal samples from 70 commercial broiler chicken flocks were examined for the presence of astroviruses, rotavirus, and reovirus by reverse transcription-polymerase chain reaction, and for the presence of group I adenovirus by polymerase chain reaction. Astroviruses were identified in 38.6% of samples tested. Both avian nephritis virus and chicken astrovirus were identified in the astrovirus positive flocks, where 74.1% of these flocks were positive for only one type of astrovirus, whereas, 25.9% of these flocks were positive for both types of astrovirus. Reoviruses, rotaviruses, and adenoviruses were identified in 21.4, 18.6, and 14.3% of these flocks, respectively. Concomitant infection with two or more viruses in the same flock were also prominent, where 5.7, 5.7, 2.9, 2.9, 1.4, and 1.4% of these flocks were positive with both astrovirus and rotavirus; astrovirus and adenovirus; astrovirus and reovirus; rotavirus and adenovirus; rotavirus and reovirus; and reovirus and adenovirus respectively. Moreover, 4.3 and 2.7% of these flocks were positive for astrovirus, reovirus, and adenovirus; and astrovirus, reovirus, and rotavirus, respectively. Further studies will focus on identifying specific viral factors or subtypes/subgroups associated with disease through pathogenesis studies, economic losses caused by infections and co-infections of these pathogens, and the costs and benefits of countermeasures.

Ornithobacterium rhinotracheale and Mycoplasma synoviae in broiler chickens in Jordan.

A cross-sectional study was conducted from November 2008 to July 2010 in commercial broiler flocks in southern (n = 50) and northern (n = 50) areas of Jordan, to determine the flock-level prevalence of Ornithobacterium rhinotracheale (ORT) and Mycoplasma synoviae (MS) infections. Tracheal swabs were collected from commercial broilers with respiratory disease and tested by polymerase chain reaction. In total, 21% (95% CI: 18-45%) and 25% (95% CI: 20-51%) of commercial broiler flocks were positive for ORT and MS, respectively. In the southern areas the prevalence of flocks with positive tracheal swabs for ORT and MS was 16% and 10%; in the northern areas the prevalence was 26% and 40%, respectively. Of the flocks tested, 7% were infected with ORT and MS simultaneously. Further epidemiological studies are recommended to determine risk factors and evaluate the economic consequences of ORT and MS infections in the region. Furthermore, studies are required to isolate ORT and MS and develop vaccines against the local field isolates.

Flock-level prevalence of Clostridium colinum in broiler flocks with digestive disease in Jordan by polymerase chain reaction.

A cross-sectional study was conducted from May to September of the year 2008 in broiler flocks in the southern and northern area of Jordan, to determine the flock-level prevalence of Clostridium colinum infection. Intestinal swabs were collected from 170 broiler flocks and tested by PCR. Among the study population, 20 flocks in both areas (11.8%, 95% confidence interval: 10 to 22%) were positive for C. colinum infection. The prevalence of positive intestinal samples in the southern and northern area of Jordan were 4.7 and 7.1%, respectively, which was statistically significant (chi(2) = 3.9 df = 1, P = 0.0482). It is recommended to conduct further epidemiologic studies to determine risk factors and to evaluate the economic consequences of the C. colinum infection in the region.

Molecular subtype of infectious bronchitis virus in broiler flocks in Jordan.

Infectious bronchitis virus (IBV) causes respiratory disease in chickens all over the world. Infectious bronchitis virus has many serotypes that do not confer cross protection against each other. The current study was designed to know which IBV types were circulating in Jordanian broiler chickens. Tracheal swabs from 175 broiler flocks at the acute phase of respiratory disease were collected. The swabs were subjected to RNA extraction and tested by reverse transcription PCR (RT-PCR). Specific-nested PCR were performed on RT-PCR products to detect and differentiate strains of Massachusetts, 4/91, and D274 types. The nucleic acid of IBV was detected in 105 out 174 (60%) broiler flocks by RT-PCR. Specific-nested PCR revealed that 35.2, 31.4, and 8.6% of these flocks had Massachusetts, 4/91, and D274, respectively, alone. In 24.8% of tested flocks, 2 types of IBV were detected. However, because the primers used in this study were designed specifically for 3 types of IBV, other types might have been present but not detected. Future work should include the isolation and molecular characterization of IBV in the region to adopt a suitable vaccination program using the common field serotypes as vaccines to protect against IBV-caused disease.

Molecular survey of avian respiratory pathogens in commercial broiler chicken flocks with respiratory diseases in Jordan.

Acute respiratory tract infections are of paramount importance in the poultry industry. Avian influenza virus (AIV), infectious bronchitis virus (IBV), Newcastle disease virus (NDV), avian pneumovirus (APV), and Mycoplasma gallisepticum (MG) have been recognized as the most important pathogens in poultry. In this study, trachea swabs from 115 commercial broiler chicken flocks that suffered from respiratory disease were tested for AIV subtype H9N2, IBV, NDV, and APV by using reverse transcription PCR and for MG by using PCR. The PCR and reverse transcription PCR results showed that 13 and 14.8% of these flocks were infected with NDV and IBV, respectively, whereas 5.2, 6.0, 9.6, 10.4, 11.3, and 15.7% of these flocks were infected with both NDV and MG; MG and APV; IBV and NDV; IBV and MG; NDV and AIV; and IBV and AIV, respectively. Furthermore, 2.6% of these flocks were infected with IBV, NDV, and APV at the same time. On the other hand, 11.3% of these flocks were negative for the above-mentioned respiratory diseases. Our data showed that the above-mentioned respiratory pathogens were the most important causes of respiratory disease in broiler chickens in Jordan. Further studies are necessary to assess circulating strains, economic losses caused by infections and coinfections of these pathogens, and the costs and benefits of countermeasures. Furthermore, farmers need to be educated about the signs and importance of these pathogens.