The Philippines is known to be one of the countries in Southeast Asia endemic for leptospirosis. However, little is known about the status of leptospirosis in this country in terms of its prevalence and incidence among humans and animals, circulating/prevailing Leptospira
species and serovars, reservoirs, transmission, and pathogenicity. The purpose of this study was to obtain information on the status of leptospirosis in the Philippines in terms of its prevalence among rats. Evidence strongly suggests that rats are the most important reservoir of leptospires. Although these animals may harbor the organisms, they do not get sick or die of leptospirosis.2
However, they become chronically infected and continuously shed the organisms for more than seven months,47
thereby contaminating the environment and making it possible for the leptospires to come in contact with other animals or humans who are exposed to this environment.2,3
Our study provided information regarding the infecting Leptospira
serovars and serogroups among rats in the Philippines on the basis of results of different tests. This study is the first published study on leptospirosis in the Philippines in which isolates were typed according to PFGE and gyrB
gene sequence analysis. The pathogenicity test of rat isolates in hamsters also provided information on the virulence of isolates from the Philippines, which was only previously reported in a study by Yanagihara and others.12
In diagnosing leptospirosis by using MAT, testing of paired serum samples is ideal for discriminate current or past infection and cross-reactions. However, in animal studies, it is often impossible to collect and test paired sera. Thus, animal studies are sometimes limited to the collection and testing of single serum samples. In our study, Leptospira
serovars that had the highest reactivity (i.e., titer) were considered to be the possible infecting serovar.45,46
However, we cannot determine if this finding is caused by past or current infections because we only had single serum samples. Furthermore, cross-reactions, especially between serogroups, are often observed in the MAT.6
However, the cross-reactivity is not observed within weeks or months after infection. Therefore, it is necessary that paired sera be tested to truly determine the infection status of subjects. The eight non-reactive rat serum samples may be negative for leptospirosis or may have antibodies against serovars that were not included in our panel.
Determining the cut-off titer (also referred to as significant titer) is another issue in the MAT.13
As previously mentioned, we were not able to detect any antibodies against Leptospira
in specific pathogen–free rats even at a reciprocal titer of 20. Therefore, we set the significant titer at 20. Nineteen of the rat serum samples had titers at this level (). Of these 19 serum samples, 16 had antibodies against multiple Leptospira
serovars. We believe that some of the antibodies with a titer of 1:20 are probably non-specific.2
In this study, leptospires isolated from rats in Metro Manila and Laguna were classified into four groups on the basis of molecular tests (, and and ). This finding suggests that these four genotypes had a strong tendency of persistence in the renal tubules. Thus, they were isolated from the kidneys of rats. It is known that upon infection, leptospires appear in the blood and are spread to other tissues and organs.2,3
They are eventually cleared by the immune response of hosts. However, they may remain in the kidney tubules and may be shed in the urine of infected hosts for weeks to more than seven months.47
There may also be a bacterial strain difference in the ability to chronically infect the hosts. In a study by Thiermann,47
Norway rats that were experimentally infected with serovars Icterohemorrhagiae and Grippotyphosa had different lengths of chronicity. Chronic infection developed faster and organisms were shed longer (220 days) in rats infected with serovar Icterohemorrhagiae than in rats infected with serovar Grippotyphosa (40 days). Another hypothesis is that the four groups of isolates in our study may be easily cultured. Thus, only these serovars and serogroups were isolated from the 46 rats but other serovars were not isolated.
As previously mentioned, one rat from Metro Manila and three rats from Laguna had mixed infections because isolates obtained from these rats had sequences and band patterns similar to serovar Manilae (for isolates that grew in Korthof's medium) and serogroup Javanica (for isolates that grew in EMJH medium). Although we do not have an evidence to support our finding, we believe that that media can be strain-selective. On the basis of our experience, L. interrogans strain Ictero No. 1 grew well in Korthof's medium but not in EMJH medium.
The MAT results showed that rats had antibodies against the four Leptospira serovars and serogroups. The MAT results of rats from Metro Manila were consistent with the molecular typing results because most antibodies detected in these rats were against serovars Manilae and Losbanos (). However, most specificities of antibodies detected in rats from Laguna were not consistent with serovars and serogroups that the rats carried (). Thus, antibodies against serovars homologous and non-homologous to the isolates were detected in rats from Metro Manila and Laguna. A possible reason for this finding may be that rats used in this study may have been previously infected with these serovars (as shown by their positive MAT results). However, because these organisms may have been already cleared from the animals, leptospires belonging to these serovars could not be isolated but antibodies remained in the rats.
MAT results of rats culture-positive for Leptospira, Metro Manila, the Philippines*
MAT results of rats culture-positive for Leptospira, Laguna, the Philippines*
Phylogenetic analysis of gyrB
has been applied to the taxonomic classification of Pseudomonas
, and Mycobacterium
gene was used to identify Leptospira
isolates from the Amami islands in Japan.26
It was also used by Slack and others38
to develop a molecular technique for identifying pathogenic Leptospira
species by conventional or real-time polymerase chain reaction amplification and sequencing of a partial fragment of the gyrB
gene. The molecular evolution of this gene was faster than that of the rrs
gene, as shown by its sequence and an average base substitution for rrs
of 1% per 50 million years; that of gyrB
at synonymous sites was 0.7–0.8% per 1 million years.51,52
This finding indicated that analysis of gyrB
was able to differentiate closely related bacterial strains, although tested strains were identical in rrs
gene sequencing. By using PFGE and gyrB
sequence analysis, we determined that isolates in our study were segregated into four groups. This finding shows that these two methods were sensitive in discriminating leptospires. Furthermore, the PFGE results identified the high clonality of isolates within the same serovars as described.7,53,54
As mentioned earlier in this report, previous seroepidemiologic studies in the Philippines had detected antibodies against several Leptospira
serovars such as Tarassovi, Sejroe, Poi, Javanica, Manilae, Losbanos, and Grippotyphosa in different human and animal serum samples.1,8–16,43
The antibodies detected in humans and animals in previous studies were also detected among the rats in our current study, which indicates that these serovars may have been present in the Philippines for several decades.
Although our sample size and sampling areas were limited, on the basis of the typing of isolates, we believe that rats in Metro Manila may be the sources of serovars Manilae, Losbanos, and Javanica, and rats in Laguna may be the sources of serogroups Grippotyphosa and Javanica. We also hypothesize that other Leptospira serovars such as Hebdomadis, Ratnapura, and Pomona, may also be prevalent in the Philippines, as shown by the presence of antibodies against these serovars in the MAT. Furthermore, isolation of serovars Manilae and Losbanos and serogroups Grippotyphosa and Javanica from rats in our study and detection of antibodies against these serovars and serogroups in humans and animals in previous studies indicate that rats may be the sources of leptospirosis transmission in the Philippines.
In terms of pathogenicity of rat isolates, hamsters that were infected with these isolates died within two weeks. The route used in this study was the intraperitoneal route, which is the most widely used infection route in most leptospiral studies reported. However, leptospires usually enter the body through cuts/wounds on the skin. It would therefore be interesting to know if the pathogenicity of the isolates would be the same using the subcutaneous route and using lower doses (i.e., less than 105). In general, injected organisms (leptospires) should be isolated from tissues or body fluids of infected animals, especially those that died from infection. However, in this study, we found that this is not always the case. As shown in , leptospires were also isolated from hamsters that were able to survive experimental leptospirosis. In contrast, leptospires were not recovered from hamsters that died after interaperitoneal injection with leptospires. This finding may show that the leptospiral doses used in our study may be sub-lethal because infected hamsters were able to survive and recover with no signs of infection. Survival of hamsters from experimental infection and the absence of recovery of injected leptospires may also indicate that isolates from the Philippines used may be non-pathogenic to the animals.
In this study, we did not definitively identify or type species of rats that we used. However, we observed that the rats obtained in Metro Manila were mostly Rattus norvegicus
and those obtained in Laguna were mostly R. tanezumi
. In previous studies, carriage of leptospirosis was found to be correlated to the species of rats and its age.3
However, we were not also able to determine the age and sex of the captured rats. It would therefore be useful if the rat species, age, and sex were identified in future investigations.
The CAAT, although time-consuming and laborious, is an indispensable method for typing Leptospira
Because of lack of resources needed for CAAT (i.e., reference antisera against Leptospira
), our laboratory was not able to perform this method. However, our use of PFGE and gyrB
sequence analysis as alternative methods for typing isolates have been done in other studies of leptospirosis.6,7,22,26,28,29,38,53
The results of our study provide useful information on the epidemiology of leptospirosis in the Philippines, which until now was not well studied. However, studies with larger sample sizes on leptospirosis among rats, humans, and other animals in other areas of the Philippines would be beneficial in determining the transmission cycle of leptospirosis and the status of this zoonosis. The observations provided in our study may also be useful in formulating leptospirosis prevention and control measures and guidelines in the Philippines and other countries with similar conditions.