Leptospira interrogans serogroup Pomona infections in the UK: is there a real threat for farm animals?

Current typing of Leptospira strains requires a combination of both serological and molecular methods. Study of the epidemiology of strains within the Pomona serogroup has proved problematic due to the limitations of serological typing methods. This is particularly true of the two main complexes within this group, namely the Pomona, Monjakov, Kennewicki cluster in Leptospira interrogans species and the Mozdok, Tsaratsovo, Kunming, Altodouro cluster in Leptospira kirschneri Strains from the south of England have been shown previously to belong to L kirschneri serovar Mozdok, strains of which are maintained by small rodents in western Europe. While they may occasionally cause disease in domestic animals, they are unlikely to be of economic importance. In contrast, L interrogans serovar Pomona type Kennewicki strains have been shown to be significant animal pathogens, especially in north America, while L interrogans serovar Pomona types Pomona and Monjakov have not been associated with significant economic loss. The purpose of this study was to examine 10 UK serovar Pomona isolates to assess type as a means of assessing the possible risk they pose to British livestock. All isolates were identified as L interrogans serovar Pomona type Pomona and were therefore unlikely to pose a significant threat to domestic animals in the UK.

Leptospira interrogans serovars Bratislava and Muenchen animal infections: Implications for epidemiology and control.

Strains of Leptospira interrogans belonging to two very closely related serovars - Bratislava and Muenchen - have been associated with disease in domestic animals, in particular pigs, but also in horses and dogs. Similar strains have also been recovered from various wildlife species. Their epidemiology is poorly understood. Two hundred and forty seven such isolates, from UK domestic animal and wildlife species, were examined by restriction endonuclease analysis in an attempt to elucidate their epidemiology. A representative sub-sample of 65 of these isolates was further examined by multiple-locus variable-number tandem repeat analysis and 22 by secY sequencing. Ten restriction pattern types were identified. The majority of isolates fell into one of three restriction endonuclease analysis pattern types designated B2a, B2b and M2a. B2a was ubiquitous and was isolated from 10 species and represented the majority of the horse and all dog isolates. B2b was very different, being isolated only from pigs, indicating that this type was maintained by pigs. The pattern M2a was reported for the majority of isolates from pigs but also was common in small rodents isolates. Five restriction pattern types were found only in wildlife suggesting that they are unlikely to pose a disease threat to domestic animals. Multiple-locus variable-number tandem repeat analysis identified six clusters. The REA types B2a and B2b were all found in one MLVA cluster while the majority of the M2a strains examined occurred in another cluster. The secY sequencing detected only one sequence type, clustered with other serovars of Leptospira interrogans.

'Leptorapide' - a one-step assay for rapid diagnosis of human leptospirosis.

SUMMARY: Leptospirosis is a globally important zoonotic infection caused by spirochaetes of the genus Leptospira. It is transmitted to humans by direct contact with infected animals or indirectly via contaminated water. It is mainly a problem of the resource-poor developing countries of the tropical and sub-tropical regions of the world but outbreaks due to an increase in travel and recreational activities have been reported in developed and more industrialized areas of the world. Current methods of diagnosis are costly, time-consuming and require the use of specialized laboratory equipment and personnel. The purpose of this paper is to report the validation of the 'Leptorapide®' test (Linnodee Ltd, Northern Ireland) for the diagnosis of human leptospirosis. It is a simple one-step latex agglutination assay performed using equal volumes of serum sample and antigen-bound latex beads. Evidence of leptospiral antibodies is determined within minutes. Agglutination is scored on a scale of 1-5 and the results interpreted using a score card provided with the kit. Validation has been performed with a large sample size obtained from individuals originating from various parts of the world including Brazil and India. The test has shown sensitivity and specificity values of 97·1% and 94·0%, respectively, relative to the microscopic agglutination test. The results demonstrate that Leptorapide offers a cost-effective and accurate alternative to the more historical methods of antibody detection.

Emergence of novel Leptospira serovars: a need for adjusting vaccination policies for dogs?

A total of 855 sera from dogs in Greece were tested for antibodies to strains belonging to the Pomona, Grippotyphosa and Australis serogroups of Leptospira to assess exposure levels to these serogroups, possible associations with clinical disease and to evaluate whether these findings support the inclusion of additional serovars in dog vaccines. Antibodies were detected in 110 (12·9%) dogs. The highest seroprevalence (4·9%) was to the proposed novel serovar Altodouro belonging to the Pomona serogroup. This serovar also showed a statistically significant association with clinical disease. Serovar Bratislava antibodies were found in 3·4% of sera. Consideration should be given to the inclusion of serovars belonging to the Pomona serogroup and serovar Bratislava in future dog vaccines for the Greek market.

Emergence, control and re-emerging leptospirosis: dynamics of infection in the changing world.

Globally, leptospirosis poses an increasing public health problem, as evidenced by markedly increasing incidence rates and multiple outbreaks in all continents. Yet, the disease is severely neglected and hence, its global burden is largely unknown. The estimated incidence of about half a million severe human cases annually is probably an underestimation while the burden for animal health is unknown. It is anticipated that current international initiatives will assess the global burden of leptospirosis, while mathematical modelling of transmission dynamics will allow the identification and testing of appropriate intervention and outbreak response measures within the coming years.

Control of canine leptospirosis in Europe: time for a change?

Changes in the formulation of the Leptospira components of dog vaccines are being considered in Europe, following changes in North America. This article discusses the options for change and recommends the continued inclusion of serovars Icterohaemorrhagiae and Canicola plus the inclusion of serovars Bratislava and Grippotyphosa (for mainland Europe only). If other serovars, such as Pomona, are to be considered in the future, then there is a need for additional clinical, cultural and serological studies across Europe to support their inclusion.

Reclassification of Leptospira parva Hovind-Hougen et al. 1982 as Turneriella parva gen. nov., comb. nov.

Analysis of the G+C content, DNA-DNA relatedness to other leptospires and 16S rRNA gene sequence of Leptospira parva showed that this species was not related to other Leptospira species. On the basis of these data, it is proposed that Leptospira parva should be transferred to the genus Turneriella as Turneriella parva gen. nov., comb. nov., with strain H(T) (=NCTC 11395(T)=ATCC BAA-1111(T)) as the type strain.

Microbiological and serological study of leptospirosis in horses at slaughter: first isolations.

A bacteriological survey of kidneys from 145 abattoir horses was performed, which resulted in the isolation of two Leptospira strains. The isolates were serologically typed as belonging to serogroups Australis and Pomona, and REA identified them as L. interrogans serovar Bratislava and L. kirschneri serovar Tsaratsovo, respectively. These are the first Leptospira isolates obtained from horses in Portugal and the Bratislava strain is the first serogroup Australis strain to be isolated in this country. The 145 horses were also serologically tested for leptospiral antibodies, and 37% had MAT titres #10878;1:10.

Detection of chlamydial antibody by fetal serology--an aid to the diagnosis of ovine abortion.

Two serological tests (indirect immunofluorescence and enzyme-linked immunosorbent assay) were developed for the detection of fetal antibody to Chlamydia psittaci. Fetal blood and thoracic fluid from 126 field cases of suspected ovine chlamydial abortion were examined using both tests. Placenta and fetal tissues (lung, liver, and kidney) from the same animals were also examined by the following conventional diagnostic methods: isolation in McCoy cells, detection of chlamydial lipopolysaccharide (LPS), modified Ziehl-Nielsen staining, and direct fluorescent antibody staining of chlamydia in frozen cryostat sections. Seventy cases were positive by fetal serology, and of these, 68 were also positive by isolation and/or LPS detection. The remaining 56 cases had negative fetal serology, and of these, 39 were positive by isolation and/or LPS detection. Results indicate that fetal serology, although less sensitive than either isolation in McCoy cells or detection of chlamydial LPS antigen, may be of particular use when placenta is not available.

Stillbirth/perinatal weak calf syndrome: a study of calves infected with Leptospira.

Leptospiral infection has been reported in calves which were either stillborn or dead from perinatal weak calf syndrome; 356 such calves were examined for evidence of associations between leptospiral infection and macroscopic, histological and microbiological findings, and the parity of the dam. Calves in which leptospiral antigen was detected in the placenta were significantly lighter by an average of 6 to 10 kg than calves with no antigen in the placenta. Calves infected with Leptospira were more likely to be infected by Actinomyces pyogenes or Bacillus species. No other significant associations were detected. The adrenal gland, lung and placenta were the most useful organs to examine for leptospiral antigen. The placenta was the only antigen-positive tissue 8.9 per cent of the calves submitted with their placenta.

Development of an ELISA to detect antibodies to a protective lipopolysaccharide fraction of Leptospira borgpetersenii serovar hardjo in cattle.

Monoclonal antibodies (Mabs) were produced against Leptospira borgpetersenii serovar hardjo-type Bovis antigens. A panel of 28 Mabs were characterised. Only the nine Mabs toward a lipopolysaccharide (LPS) fraction of 18, 24 kDa bands and a 26-28 kDa smear showed agglutinating, leptospiricidal and growth-inhibition activities, and passively protected hamsters against renal infection with hardjo. They also reacted strongly in the CH-ELISA, captured killed whole hardjo leptospires, gave good fluorescence in indirect FAT against smears of hardjo culture and exhibited no cross reactivity with strains in heterologous serogroups. On the basis of optimal activity in a range of tests, one IgG class Mab (designated 25) was selected for use in an antibody-capture ELISA system for the detection of bovine anti-hardjo antibodies. The system gave a wide separation of absorbance values between positive and negative sera at a 1:10 dilution. The antibodies detected by this assay are believed to be protective anti-LPS IgG.

Development of an immunomagnetic antigen capture system for detecting leptospires in bovine urine.

A magnetic bead antigen capture system which combined the use of two evolving techniques - immunomagnetic separation (IMS) and time-resolved fluoroimmunoassay (TR-FIA) - was developed to detect Leptospira borgpetersenii serovar hardjo in bovine urine. The assay utilised monoclonal antibody coated magnetic beads to capture leptospiral antigen which was in turn detected using another monoclonal antibody (Indicator) labelled with biotin. Signal was generated by the binding of europium labelled streptavidin to indicator antibody. The sensitivity of the assay was improved from 10(3) to 10(2) leptospires per ml by using an ethanol precipitation procedure to treat each sample. The assay detected only 31 of 56 (55 per cent) urine specimens culture-positive for hardjo, but seven of 24 urine samples culture-negative for hardjo were identified as positive by the assay. These seven samples were from animals which were culture positive on at least one other occasion. These results suggest that this system should be further investigated as a complementary test to culture for the identification of hardjo carrier animals.

Experimental reproduction of iodine deficiency in cattle.

The role of iodine deficiency in stillbirth/perinatal weak calf syndrome was investigated in pregnant heifers. Five heifers were fed an iodine deficient diet (mean [sd] iodine concentration 0.06 [0.01] mg/kg dry matter [DM]) and six received an iodine sufficient diet (mean [sd] iodine concentration 1.45 [0.27] mg/kg DM). The diets consisted of wheat and soyabean meal with added minerals and vitamins (with or without iodine) and were fed to the heifers over the final four to five months of pregnancy. The iodine deficient diet produced clinicopathological changes and pathological changes in the thyroid glands of both the heifers and their offspring. However, all the calves in the iodine deficient group were born clinically normal.

Magnetic immuno capture PCR assay (MIPA): detection of Leptospira borgpetersenii serovar hardjo.

Magnetic immuno PCR assay (MIPA) was developed for the rapid detection of leptospires excreted in urine samples (n = 59) collected from 35 experimentally infected cattle. The immunomagnetic separation of leptospires from inhibitors in frozen formalin fixed bovine urine prior to PCR detection resulted in a marked improvement on previous detection methods. MIPA is a rapid 5 step protocol requiring 70 mins preparation time prior to amplification, which consistently detects 10(1) organisms. MIPA detected 76% (38/50) of culture positive urines and in addition three urines that were culture negative were shown to be positive by this method of detection. Consequently we conclude that whilst MIPA is an improvement on previously published PCR detection methods, the culture of the organism is still the standard against which other detection methods have to be compared.