Identification and genetic characterisation of Giardia and Cryptosporidium strains in humans and dairy cattle in the Waikato Region of New Zealand.

Giardia intestinalis and Cryptosporidium parvum are commonly acquired waterborne parasites but little is known about their transmission cycles with regard to humans and animals. Faecal samples were collected over two time periods within the Waikato region of New Zealand from dairy cattle and humans and all positive isolates were genotyped. Of the 724 faecal specimens examined (354 cows, 304 calves and 66 humans), 16 cows, 32 calves and 5 humans were positive for G. intestinalis. Phylogenetic group 1 was found in 26 G. intestinalis positive dairy cattle with 22 being group 2. One G. intestinalis positive human isolate was group 1 and four were group 2. Of the 724 faecal specimens examined two cows, 33 calves and 66 human specimens were positive for C. parvum. All 35 C. parvum positive dairy cattle exhibited the bovine genotype while the 66 positive humans showed a seasonal shift in the prevalent genotype with the bovine dominating the spring (100%) and the human dominating the late summer period (96%).

Prevalence and strain differentiation of Giardia intestinalis in calves in the Manawatu and Waikato regions of North Island, New Zealand.

Giardia intestinalis has been reported in newborn calves world-wide; however, information on the extent of G. intestinalis in New Zealand calves has to date been very limited. The current study attempted to establish the prevalence rate of G. intestinalis in calves up to 8 weeks old in New Zealand. More than 700 calf fecal specimens were collected during the spring calving seasons of 1998 and 1999 from two regions in North Island, New Zealand (Manawatu and Waikato) and tested for the presence of G. intestinalis. In addition to determining the presence of G. intestinalis in newborn calves, sequence analysis was performed using specific amplification primers developed to target a section of the ribosomal DNA (rDNA). This locus is considered to be rapidly evolving, and therefore, suitable for use in the elucidation of phylogenetic relationships between G. intestinalis isolates. Sequencing was performed using G. intestinalis DNA extracted from cysts collected directly from the calf fecal matter. There was no culturing of the G. intestinalis isolates either in vivo or in vitro. Over 40% of all collected calf fecal specimens contained G. intestinalis cysts and rDNA sequence analysis revealed two different sequences among calf isolates. These sequence differences were not found to correspond to a particular season, geographical region or farming practice. Preliminary phylogenetic analysis suggests that these two rDNA sequence types are indicative of calf hosts.

Chronic non-progressive pneumonia of sheep in New Zealand - a review of the role of Mycoplasma ovipneumoniae.

Chronic non-progressive pneumonia (CNP) is a common disease which affects lambs in New Zealand during late summer and autumn. Mycoplasma ovipneumoniae can be recovered from a high proportion of lesions but it is also present in some normal lungs. Bacteria, especially Pasteurella haemolytica, can also be recovered from more than half the lungs of affected animals. Isolates of M. ovipneumoniae are genetically heterogeneous, as demonstrated by examination of their DNA or total cellular proteins, and are serologically heterogeneous as shown by metabolic inhibition tests. The number of strains present in New Zealand is large and several distinguishable strains can be recovered from each affected lung. Mycoplasma ovipneumoniae has pathogenic potential as indicated by its ability to produce hydrogen peroxide, cause ciliostasis and by its possession of a capsule. Chronic non-progressive pneumonia can be transmitted consistently to over 50% of lambs by inoculation of pooled pneumonic lung homogenate and transmission can be suppressed by broad spectrum antibiotics. In contrast, penicillin does not prevent the development of lesions but diminishes their severity. Pooled lung homogenate treated with digitonin, which inactivates mycoplasmas, has failed to transmit CNP. Pure cultures of M. ovipneumoniae produce only mild lesions in some animals, whereas inoculation with pooled lung homogenate (from which no viruses were isolated) containing mixed strains of M. ovipneumoniae and free from bacteria, is more effective in producing lesions. Research work to date suggests that CNP may be initiated by colonisation of the lung by M. ovipneumoniae which causes ciliostasis and elicits an exudate allowing colonisation of the lungs by bacteria especially M. haemolytica and by other strains of M. ovipneumoniae. The immune response to the initial strain of M. ovipneumoniae may inhibit its replication but would be less effective in inhibiting heterologous strains of the organism allowing their sequential replication. Eventually production of a broad immune response to M. ovipneumoniae would lead to its elimination which in turn would facilitate the elimination of other microorganisms and the resolution of lesions. As natural immunity to CNP occurs within the first year, it may be possible to develop an effective and useful vaccine. Such a vaccine may need to include multiple strains of M. ovipneumoniae.

A study of the heterogeneity of isolates of Mycoplasma ovipneumoniae from sheep in New Zealand.

To investigate the heterogeneity of Mycoplasma ovipneumoniae, sixty isolates from three sheep on each of twenty farms were examined by restriction endonuclease analysis (REA) and SDS-PAGE. All were found to be different except for three isolates obtained from one farm. The protein and REA patterns of individual isolates were both highly reproducible and remained unchanged following long term passage (approximately 400 generations) in vitro. No plasmids were detected in the twelve strains which were examined and when two isolates were co-cultured in vitro, no genetic interchange, as judged by changes in REA patterns were detected. Since the heterogeneity of M. ovipneumoniae when examined by SDS-PAGE is too great to allow groups to be recognised, it could be advantageous for this purpose if only surface proteins were compared. As a preliminary step to this end we have identified several surface proteins of M. ovipneumoniae and found that some are common to all strains, one surface protein was shared by five of the eight strains examined and another was unique to one strain. This approach has the potential to allow the recognition of grouping of M. ovipneumoniae isolates.

The isolation of multiple strains of Mycoplasma ovipneumoniae from individual pneumonic sheep lungs.

The heterogeneity of Mycoplasma ovipneumoniae isolates from the lungs of sheep with chronic non-progressive pneumonia (CNP) from the same flock raised the possibility that multiple isolates derived from one lung were not all identical. To test this hypothesis, thirty isolates were obtained from each of six pneumonic sheep lungs at slaughter. Four lungs had relatively severe lesions and from each of these, three or four strains of M. ovipneumonia, distinguishable by REA and in most cases by SDS-PAGE, were detected. From the lungs of each of two sheep with mild lesions, two strains of M. ovipneumoniae were detected. Four isolates from one lung were further examined by restriction endonuclease analysis (REA) using many restriction endonucleases. Those which differed with EcoRI also differed when other restriction endonucleases were used. However, partial digests occurred mainly with those restriction endonucleases which recognise cytosine-rich sequences. The presence of multiple strains of one species of microorganism in individual lesions is an unusual concept which may not be limited to one disease or to one host.

Colonisation of the respiratory tract of lambs by strains of Mycoplasma ovipneumoniae.

The age and time of year when colonisation of the nasal cavity of lambs by Mycoplasma ovipneumoniae occurs; the persistence of the organism, and its prevalence in the lungs at slaughter were examined in 2 flocks of sheep in New Zealand. No colonisation had occurred at the time of weaning at 6-7 weeks, but M. ovipneumoniae was recovered from most lambs on at least one occasion before they were slaughtered when about 8 months old. In most cases, colonisation of the nasal cavity by M. ovipneumoniae was a transient phenomenon. At slaughter M. ovipneumoniae was recovered from the lungs of 89% of the lambs of one flock and 80% of the other flock. Bacterial restriction endonuclease DNA analysis (BRENDA) of 34 nasal isolates from one flock showed that it was possible to identify 7 "groups" each with markedly different BRENDA patterns. Lambs initially colonised by one strain, often lost that strain, and if recolonisation occurred it was with a different strain. M. ovipneumoniae was recovered at slaughter from the lungs of most lambs, both normal and pneumonic. The isolates from one flock were examined by BRENDA, and approximately 90% of them gave similar or identical patterns. The predominant strain isolated from the lungs had been recovered from the nasal cavity of many of the lambs about 3 weeks earlier. This suggests that the nasal and lung isolates do not represent independent populations. However, nasal strains may differ in their ability to colonise the lungs.(ABSTRACT TRUNCATED AT 250 WORDS)

Comparison of Mycoplasma ovipneumoniae isolates using bacterial restriction endonuclease DNA analysis and SDS-PAGE.

Sixteen isolates of Mycoplasma ovipneumoniae recovered from the nasal tract or lungs of sheep from different flocks in New Zealand were examined by bacterial restriction endonuclease DNA analysis (BRENDA) using EcoR1 and by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). All isolates gave BRENDA patterns which differed entirely from one another. Following 20 serial passages (corresponding to approximately 67 generations) of an isolate, no change was detected in the BRENDA pattern. When eight isolates were examined by SDS-PAGE most bands were common but, nevertheless, each isolate was unique in the sense that they differed from one another in one or more bands. The marked heterogeneity of patterns observed when strains of M. ovipneumoniae are compared by BRENDA, together with the stability of such patterns over many generations, will enable this approach to be used to study the epidemiology of individual strains of M. ovipneumoniae within a flock.