Joan A. Smyth

Nucleotide sequence analysis of a novel circovirus of canaries and its relationship to other members of the genus Circovirus of the family Circoviridae

The circular, single-stranded DNA genome of a novel circovirus of canaries, tentatively named canary circovirus (CaCV), was cloned and sequenced. Sequence analysis indicated that the genome was 1952 nucleotides (nt) in size and had the potential to encode three viral proteins, including the putative capsid and replication-associated (Rep) proteins. The CaCV genome shared greatest sequence similarity (58.3% nt identity) with the newly characterized columbid circovirus (CoCV) and was more distantly related to the two porcine circovirus strains, PCV1 and PCV2, beak and feather disease virus (BFDV) and a recently isolated goose circovirus (GCV) isolate (46.8-50.9% nt identity). In common with other members of the Circovirus genus, several nt structures and amino acid motifs thought to be implicated in virus replication were identified on the putative viral strand. Phylogenetic analysis of both the capsid and Rep protein-coding regions provided further evidence that CaCV is more closely related to CoCV and BFDV and more distantly related to GCV, PCV1 and PCV2.

Study of leg weakness in two commercial broiler flocks

The major causes of leg weakness/lameness were investigated in two male commercial broiler flocks. The numbers of dead and lame birds culled from the flocks each day were recorded by the flock managers. Forty-four lame birds and 22 sound birds were examined postmortem during a period of six weeks and the proximal and distal end of each femur, tibiotarsus and tarsometatarsus were examined histologically. Attempts were made to isolate bacteria and viruses from the proximal end of each femur. Blood samples were examined for antibodies to chicken anaemia virus (CAV), infectious bursal disease virus (IBDV) and Mycoplasma species. Bacterial chondronecrosis with osteomyelitis was identified in the proximal end of the femur of eight of the 44 lame birds, and in the proximal end of the tibiotarsus of a further bird (20.4 per cent). Gram-positive bacteria were present in all the lesions. Staphylococcus aureus was recovered from 62.5 per cent of the lesions confirmed by histology. Bacterial chondronecrosis associated with S aureus has thus been identified as an important cause of leg weakness in these commercial broilers. Lesions suggestive of the condition were visible macroscopically in only 11.1 per cent of the cases subsequently diagnosed by histology and bone histology is therefore required before a diagnosis can be excluded. Angular limb deformities (13.6 per cent) and spondylolisthesis (11.4 per cent) were the most common macroscopic lesions identified as causes of lameness. The overall incidence of tibial dyschondroplasia was similar in both the lame and sound broilers, but severe lesions were found only in lame birds (4.5 per cent).

Prevalence of netb among some clinical isolates of Clostridium perfringens from animals in the United States.

A previously unknown pore forming toxin, called NetB toxin, which is produced by some Australian strains of Clostridium perfringens has recently been reported. This toxin was reported to be critical to the development of the disease necrotic enteritis, in chickens. To investigate the occurrence of the toxin gene (netB) in non-Australian C. perfringens strains, one hundred and six American isolates of C. perfringens were examined. Ninety-two isolates were from chickens, and 14 were from cattle. The netB gene was found in 14 isolates from chickens (7 from chickens with necrotic enteritis, and 7 from unrelated chickens with no evidence of necrotic enteritis). The netB gene was also detected in an isolate recovered from a 3-year-old cow with liver abscesses. The products of all positive netB PCR reactions were sequenced, and these showed 100% nucleotide identity to the netB sequence published in GenBank. Five isolates which had been recovered from five chickens with necrotic enteritis (from four flocks) were netB negative. An additional 24 isolates recovered from one of these lesioned chickens were also netB negative. The present study represents the first study of C. perfringens isolates outside Australia for netB, and the first identification of netB in an isolate from a species other than chickens. The results indicate that the role of NetB in the induction of necrotic enteritis needs to be further investigated, by determining the disease producing capability of both netB positive strains recovered from normal chickens, and netB negative strains recovered from chickens with necrotic enteritis.

A Lutein-Enriched Diet Prevents Cholesterol Accumulation and Decreases Oxidized LDL and Inflammatory Cytokines in the Aorta of Guinea Pigs

Lutein has been shown to be protective against age-related macular degeneration; however, the antiinflammatory and antioxidant effects of this carotenoid in aortas are less known. Guinea pigs were fed a hypercholesterolemic diet (0.25 g cholesterol/100 g) and randomly allocated to a control group (n = 9) or a lutein group (n = 10) (0.01 g/100 g lutein) [corrected] and fed the experimental diets for 12 wk. Plasma LDL cholesterol and TG did not differ between groups; however, the lutein group had lower concentrations of medium size LDL (P < 0.05). As expected, guinea pigs from the lutein group had higher concentrations of plasma and liver lutein than those from the control group (P < 0.0001). Aortic cholesterol and malondialdehyde concentrations were lower in the lutein group (9.6 ± 2.8 mmol/g and 1.69 ± 1.35 nmol/mg protein) compared to the control group (15.5 ± 2.3 mmol/g and 2.98 ± 1.45 nmol/mg protein) (P < 0.05). Hematoxilin and eosin staining indicated that aortas from the control group presented focal intimal thickening, whereas either less thickness or no visible thickness was present in aortas from the lutein group. Oxidized LDL (oxLDL) was lower both in plasma and aorta in the lutein group compared to the control group (P < 0.001). Aortic cytokines were also lower in the lutein group (P < 0.05). Plasma lutein and oxLDL (r = -0.79; P < 0.0001) and plasma lutein and aortic oxLDL (r = -0.64; P < 0.0001) were negatively correlated. These data suggest that lutein exerts potent antioxidant and antiinflammatory effects in aortic tissue that may protect against development of atherosclerosis in guinea pigs.

Nucleotide sequence-based identification of a novel circovirus of canaries.

Circovirus-like, spherical particles measuring 16 to 18 nm in diameter were detected in organ homogenates from adult canaries that had died after a short illness characterized by dullness, anorexia, lethargy and feather disorder. A polymerase chain reaction method, based on degenerate primers specific to conserved amino acid sequences in the circovirus replication-associated protein, was used to amplify DNA specific to a novel circovirus, tentatively named canary circovirus (CCV). Sequence analysis of a 510 nucleotide genomic fragment indicated that CCV exhibited 67.4, 64.9, 53.7 and 53.9% nucleotide identities and 70.0, 61.8, 40.4 and 40.1% amino acid identities with columbid (pigeon) circovirus (CoCV), beak and feather disease virus (BFDV), porcine circovirus type 1 and porcine circovirus type 2, respectively. CCV therefore represents a new avian virus of the genus Circovirus of the family Circoviridae, and is more closely related to CoCV than BFDV.The availability of nucleotide sequence data will facilitate the development of DNA-detecting diagnostics with which the prevalence of CCV infections can be assessed.

Disease producing capability of netB positive isolates of C. perfringens recovered from normal chickens and a cow, and netB positive and negative isolates from chickens with necrotic enteritis

Necrotic enteritis is a serious disease of chickens and turkeys caused by Clostridium perfringens. Recently, a pore forming toxin of C. perfringens, called NetB, was reported and suggested to be critical to the development of necrotic enteritis. To investigate further the importance of NetB in the development of necrotic enteritis, toxin production and disease producing ability of (1) netB positive isolates recovered from normal chickens, (2) a netB positive isolate recovered from a cow, (3) netB negative isolates recovered from chickens with necrotic enteritis and (4) netB positive isolates recovered from chickens with necrotic enteritis, were examined. None of the netB negative isolates recovered from chickens with necrotic enteritis produced disease in challenged chickens. All netB positive isolates produced necrotic enteritis in challenged chickens, although there were substantial differences in the incidence and severity of lesions. Thus, one netB isolate produced severe lesions in 80% of challenged chickens, while another produced lesions in only 20% of challenged chickens, and these were very mild. The netB positive isolate from a cow, produced lesions in 90% of challenged chickens with severe lesions in 50%. While these findings would generally support the concept that netB is very important to development of necrotic enteritis, the finding that there was a wide range of virulence among the netB positive isolates suggests that other critical factors are also involved. This study has also demonstrated for the first time that C. perfringens strains from a mammalian species and from normal chickens, can cause necrotic enteritis in chickens.

Development of an experimental model of bacterial chondronecrosis with osteomyelitis in broilers following exposure to Staphylococcus aureus by aerosol, and inoculation with chicken anaemia and infectious bursal disease viruses

A series of experiments was designed in an attempt to reproduce bacterial chondronecrosis with osteomyelitis in broiler chickens using a natural route of infection. Birds in isolators were exposed to a suspension of Staphylococcus aureus by aerosol or exposed to S. aureus and subsequently inoculated with chicken anaemia virus (CAV) alone, or with CAV and infectious bursal disease virus (IBDV). Subsequently, S. aureus was recovered and bacterial chondronecrosis with osteomyelitis was diagnosed, by histology, in the proximal end of the femur and/or tibiotarsus of lame birds exposed to S. aureus with and without CAV and IBDV infections. Birds fed 60% of the recommended feed intake for the breed developed a lower incidence of S. aureus infection and/or bacterial chondronecrosis (P < 0.05) than birds fed 100% of the recommended intake. A significantly lower incidence of S. aureus was recovered (P < 0.05) in birds simultaneously exposed to S. aureus and inoculated with CAV and IBDV at day 21, than in birds exposed to S. aureus at day 10, and inoculated with CAV and IBDV at day 21. With the exception of birds exposed to S. aureus at 1 day old, a higher incidence of bacterial chondronecrosis was diagnosed in birds exposed to S. aureus and inoculated with CAV and IBDV than in birds exposed to S. aureus alone. It is hypothesised that inoculation with CAV and IBDV at day 21 enhanced the development of bacterial chondronecrosis in birds exposed to S. aureus at day 10 and fed 100% of the recommended feed intake or ad libitum.

Comparison of Staphylococcus aureus recovered from personnel in a poultry hatchery and in broiler parent farms with those isolated from skeletal disease in broilers

Personnel from one broiler hatchery, and workers on 18 separate broiler parent farms which supply the hatchery, were tested for hand and nasal carriage of Staphylococcus aureus. In both locations, nasal carriage of S. aureus was more common than hand carriage. A total of 63 S. aureus strains were characterised by biotyping, protein A analysis and pulsed field gel electrophoresis (PFGE) typing. Of these, 36 were recovered from broiler hatchery personnel, 14 from broiler parent farm personnel and 13 from cases of skeletal disease in commercial broilers. Biotyping and protein A analysis indicated that none of the strains recovered from hatchery personnel were of the poultry biotype, but that two strains recovered from the hands of two broiler parent farm personnel could be grouped together with 12/13 of strains recovered from skeletal disease in broilers, as poultry biotypes. PFGE-typing could not distinguish 9/13 strains recovered from skeletal disease in broilers and one of the strains from the broiler parent farm personnel from isolate 24 (I. 24), which is the predominant S. aureus strain type associated with clinical disease in N. Ireland broiler flocks. The present study found no evidence of nasal carriage of S. aureus strains of poultry biotype by humans. The finding of hand carriage by broiler parent farm personnel, suggests that handling by personnel may contribute to the dissemination of I. 24 or other S. aureus strains associated with skeletal disease in broilers.

Evaluation of polymerase chain reaction and dot blot hybridisation tests in the diagnosis of pigeon circovirus infections.

Polymerase chain reaction (PCR) and dot blot hybridisation (DBH) tests for detecting pigeon circovirus (PiCV) DNA were developed and evaluated using tissue samples obtained from diseased and clinically normal pigeons, which originated in Belgium and Northern Ireland. When PCR product was visually detected, the limit of detection of the PCR test was 31 fg, while that of the DBH was 1.6p g. For evaluation purposes, the results of the PCR and DBH tests, performed with DNAs extracted from samples of bursa of Fabricius (BF), were compared with those of in situ hybridisation (ISH) and histology. In 32 samples tested by all four tests, 27 (84%) were positive by PCR, 24 (75%) were positive by ISH, 20 (63%) were positive by DBH, and 13 (41%) were positive by histology. Additional PCR testing showed that in some disease-affected birds, PiCV DNA could be detected in a range of tissues including thymus, spleen, liver, kidney and brain. The PCR detection of PiCV DNA in BF samples from clinically normal birds indicated that PCR can detect infections in the absence of disease, a finding that mitigates against its use as a disease diagnostic. In addition, nucleotide sequence determinations indicated that PCR test performance was adversely affected by the sequence diversity exhibited by selected PiCVs. The application of the DBH test to dilutions of test samples indicated that the BF from some diseased pigeons contained very large amounts of virus DNA, as much as 10(13)genome copies/g tissue, and suggested that this test may be a convenient method of providing a semi-quantitative estimate of virus load.

Detection of Circovirus Infection in Pigeons by in Situ Hybridization Using Cloned DNA Probes

Degenerate primers were designed based on known sequence information for the circoviruses psittacine beak and feather disease virus and porcine circovirus and applied by polymerase chain reaction (PCR) to known virus-infected bursa of Fabricius (BF) from a pigeon. A 548–bp DNA fragment was amplified and shown to be specific to a novel circovirus, named pigeon circovirus (PiCV), and was used to produce sensitive and specific probes for detection of circovirus DNA by in situ hybridization (ISH). Using ISH on BF from 107 pigeons submitted for necropsy, infection was detected in 89%, compared with a histologic detection rate of 66%. Using the ISH technique, infected cells were also found in liver, kidney, trachea, lung, brain, crop, intestine, spleen, bone marrow, and heart of some birds. Large quantities of DNA were present in some of these tissues, and in the absence of BF, liver in particular is identified as a potentially useful organ to examine for presence of PiCV. This high prevalence of infection in diseased birds is noteworthy, emphasizing the need for studies to determine the precise role of this virus as a disease-producing agent.