Janet M. Bradbury

Demonstration of sites of latency of infectious laryngotracheitis virus using the polymerase chain reaction.

Mature laying chickens were inoculated intratracheally with a field strain of infectious laryngotracheitis (ILT) virus. Tracheal swabs were collected regularly from all birds for virus culture. At various times post-inoculation, pairs of birds were killed and tissues removed for detection of virus products using conventional tissue homogenization and culture, organ culture, indirect immunofluorescence (IF) and also the polymerase chain reaction (PCR). The latter was used to detect a DNA sequence from the ILT virus thymidine kinase gene. Following inoculation the birds developed mild respiratory disease with clinical signs characteristic of ILT from 3 to 10 days post-inoculation. Trachea and turbinate tissues were virus-positive as determined by virus isolation, organ culture, IF and PCR on day 4 post-inoculation. After recovery from the acute phase, virus shedding initially ceased, then intermittent, low level shedding was recorded for five of the six remaining birds. In an attempt to locate sites of latency, pairs of birds were sampled at 31, 46 and 61 days post-inoculation. Virus was not detected in upper respiratory tract or ocular tissues by conventional techniques, or in the trigeminal, proximal and distal ganglia. All tissues were also negative by PCR, except for the trigeminal ganglia of five of the six birds. All PCR-positive birds had previously shed ILT virus intermittently between days 19 and 59 post-inoculation. As we did not detect viral DNA in any of the other tissues sampled from clinically recovered birds, we conclude that the trigeminal ganglion is the main site of latency of ILT virus.

Latency and reactivation of infectious laryngotracheitis vaccine virus.

SummaryLatency and reactivation of a commercial infectious laryngotracheitis virus vaccine were demonstrated in live chickens. Virus was re-isolated at intervals between seven and fourteen weeks post-vaccination and this may be of epizootiological significance.

Mycoplasma imitans sp. nov. is related to Mycoplasma gallisepticum and found in birds

A mycoplasma designated strain 4229T (T = type strain) was isolated in 1984 from the turbinate of a duck in France, and similar strains were isolated from geese in France and from a partridge in England. All of these strains were originally identified as Mycoplasma gallisepticum by immunofluorescence and growth inhibition tests, but subsequent serological and molecular studies indicated only a partial relationship to this species and DNA-DNA hybridization studies revealed only approximately 40 to 46% genetic homology with M. gallisepticum PG31T. In this study morphological, cultural, and physical investigations were carried out on strain 4229T and partridge strain B2/85, after we first demonstrated the similarity between these organisms by performing a restriction enzyme analysis of their DNAs. Both strains had phenotypic properties very similar to M. gallisepticum properties, including the presence of an attachment organelle. As a result of these investigations, the organisms were assigned to the class Mollicutes, the order Mycoplasmatales, and the genus Mycoplasma. They fermented glucose, reduced triphenyl tetrazolium chloride aerobically and anaerobically, and exhibited hemadsorption and hemagglutination, but other biochemical tests were negative. Apart from a serological cross-reaction with M. gallisepticum, these organisms exhibited no significant relationship with any previously described Mycoplasma species as determined by growth inhibition or immunofluorescence tests or with a number of additional serovars and unclassified avian strains. This Mycoplasma taxon therefore appears to be a new species, for which we propose the name Mycoplasma imitans. The type strain is strain 4229 (= NCTC 11733 = ATCC 51306).(ABSTRACT TRUNCATED AT 250 WORDS)

Exacerbation of Mycoplasma gallisepticum infection in turkeys by rhinotracheitis virus.

Groups of 1-day-old turkey poults from a parent flock free of antibodies to turkey rhinotracheitis virus (TRTV) and the pathogenic mycoplasmas, were infected by eyedrop with virulent TRTV, with Mycoplasma gallisepticum (Mg) or with both agents together. Dual infection resulted in increased morbidity compared with those groups given single infections. The presence of the Mg in the dual infection had no apparent effect on the pathogenesis of the virus, but the virus caused the Mycoplasma to be more invasive. Mg infection caused a transient depression in TRTV ELISA antibody titres at 29 days post-inoculation. At 14 days post-infection Mg haemagglutination inhibition (HI) and rapid serum agglutination (RSA) titres were higher (P <0.01) in the mixed infection group compared with those infected with Mg alone, but there was no significant difference between ELISA antibody titres of these two groups.

Studies on the adsorption of certain medium proteins to Mycoplasma gallisepticum and their influence on agglutination and haemagglutination reactions

Serum proteins from Mycoplasma gallisepticum culture medium could be detected on the organisms as a result of incubation at low pH. Only certain of the serum proteins, including IgG and IgM, were found, and the adsorption appears to depress the haemagglutinating activity of the organisms. There was no obvious effect on slide agglutination (SA) sensitivity but an incidental finding was that brief acid treatment enhanced the SA sensitivity of an antigen prepared from a young culture.

Investigations into the survival of Mycoplasma gallisepticum, Mycoplasma synoviae and Mycoplasma iowae on materials found in the poultry house environment.

Following preliminary experiments to determine suitable methods for studying mycoplasma survival, suspensions of Mycoplasma gallisepticum (four strains), Mycoplasma synoviae (two strains) or Mycoplasma iowae (two strains) were seeded onto replicate samples of cotton, rubber, straw, shavings, timber, food, feathers and human hair. The organisms were also seeded onto human skin, ear and nasal mucosa. All samples were cultured for viability after 4, 8, 12 and 24 h, and then daily up to 6 days. The identity of recovered mycoplasmas was confirmed by indirect immunofluorescence. All three Mycoplasma species survived for the longest time on feathers with M. gallisepticum surviving between 2 and 4 days and M. synoviae 2 to 3 days. The type strain of M. iowae remained viable for 5 days on feathers, while the field strain was still viable at the end of the 6-day experiment. This strain also survived for at least 6 days on human hair and several other materials. M. gallisepticum survived on human hair up to 3 days and one recent field isolate also survived in the nose for 24 h. Survival times of the organisms were generally less on other materials although M. gallisepticum could be isolated from straw, cotton and rubber samples after 2 days.

Genome isomerism in two alphaherpesviruses:Herpesvirus saimiri-1(Herpesvirus tamarinus) and avian infectious laryngotracheitis virus

SummaryGenome isomerism of avian infectious laryngotracheitis virus (ILT) andHerpesvirus saimiri-1 (HVS-1) (formerlyH. tamarinus) was investigated using restriction endonuclease analysis and scanning densitometry. ILT DNA was found to have 2 isomers with a molecular weight of 109×106. HVS-1 DNA was found to have four isomers with a molecular weight of 102×106.

Poultry mycoplasmas: sophisticated pathogens in simple guise

1. Mycoplasmas are a genus within the class Mollicutes (trivial name mollicutes), which are the smallest known prokaryotes capable of self-replication. They have a very small genome, and have evolved to this ‘minimalist’ status by losing non-essential genes, including those involved in cell wall synthesis. 2. The mollicutes exploit their limited genetic material to the maximum and many are successful pathogens in man, animals, birds and plants. Most of those of veterinary importance are in the genus Mycoplasma and include 4 poultry pathogens of economic importance: Mycoplasma gallisepticum, Mycoplasma synoviae, Mycoplasma meleagridis and Mycoplasma iowae. 3. The pathogenetic mechanisms of mycoplasmas are not fully understood, but they are successful pathogens because they can enter the host and multiply, evade the defence mechanisms, cause damage and escape to infect new hosts. 4. M. gallisepticum is one of several motile species and possesses a terminal tip structure that mediates adherence to its target tissues. For some species, including M. gallisepticum, some of the organisms may become intracellular. 5. Some Mycoplasma species, including the pathogenic poultry species, have a remarkable ability to vary their major surface antigens, a mechanism that is thought to help them to persist in their host by evading the immune response. 6. The molecular and cellular events that lead to the development of lesions and clinical disease are still obscure. Some lesions appear to be the result of indirect damage from the hosts inflammatory and cellular responses. 7. Despite short survival times in the environment, mycoplasmas are able to transmit successfully to new hosts. In poultry flocks there is both horizontal and vertical transmission, the former being encouraged by intensive husbandry and stress factors. Establishing the pathways of transmission and the possible role of other birds, such as game and wild birds, as intermediate vectors between poultry flocks is now greatly aided by the availability of modern molecular methods for strain typing.

Restriction Endonuclease Patterns of Some European and American Isolates of Avian Infectious Laryngotracheitis Virus

Eleven isolates of infectious laryngotracheitis virus (nine European and two American) were compared by restriction endonuclease analysis of their DNA after radiolabeling with 32P. Digestion with KpnI gave identical cleavage patterns for all the European isolates, but the two American viruses (one field and one vaccine) showed some differences from them and from each other. In the case of the American vaccine strain, however, these differences were only minor. After BamHI digestion, only the American field isolate appeared to be different, whereas with HindIII, all 11 isolates were identical.

Development and evaluation of an improved diagnostic PCR for Mycoplasma synoviae using primers located in the haemagglutinin encoding gene vlhA and its value for strain typing

Using published primers, detection of Mycoplasma synoviae and strain identification using the vlhA gene sequence was attempted. However, of 21 M. synoviae strains examined, three could not be amplified, so a new reverse primer was designed with a target in the conserved region of the vlhA gene. This allowed all 21 M. synoviae strains, a further nine strains and also material from 11 swab samples from M. synoviae-positive birds, to produce a PCR product, suggesting that the method could also be suitable for clinical specimens. The protocol was then tested on the type strains of M. synoviae and the other 22 recognised avian Mycoplasma species, with amplification of M. synoviae only. Further testing demonstrated that this PCR was equally or more sensitive than other PCR tests used to detect M. synoviae. Subsequent DNA sequence analysis of the PCR product based on percent similarity and evolutionary relationship appeared to be a useful tool for strain differentiation.