Leptospira spp. can be found worldwide regardless of climate and has been presumed to be the most widespread zoonoses . Despite it being common, the diagnosis of canine leptospirosis is not often made unless the dog was presented with clinical manifestations such as fever, jaundice, renal and/or liver failure. Clinical diagnosis remains a challenge but with the aid of confirmatory laboratory tests, a diagnosis can be derived to allow immediate administration of therapeutic regime  especially for dogs diagnosed with leptospirosis. This study demonstrated the utility of direct detection using serological and molecular methods followed by bacterial isolation in dogs with kidney and/or liver disease and found that dogs can shed Leptospira spp., further contaminating the environment and poses a risk of infection to their owners.
Microscopic agglutination test is a sensitive assay, but because of the antigenic heterogeneity of Leptospira spp., the test requires many serovars as antigens . The overall serological detection of leptospiral infection in dogs with kidney and/or liver disease was 42.7%, much higher compared to the previous studies locally. The reason could likely be due to the specific selection of recruited dogs and serum tested against 20 leptospiral serovars selected. In comparison, previous studies investigated a larger population of apparent healthy shelter and working dogs , some investigated healthy dogs from a single location [21, 22] and one study was carried out among the pet dogs . All these studies only aimed to determine seropositive among apparently healthy dogs using a panel of 10 leptospiral serovars, unlike in this study.
The MAT titre frequently observed were at 1:100 (n = 21), and perhaps could be used as a potential cut-off titre for diagnosis of leptospirosis as these dogs were clinically sick with the supportive evidence of elevated kidney and/or liver profiles. Alarmingly, three dogs with a titre level of 1:100 did not survive despite being treated and serovar Bataviae was detected. These three dogs showed clinical signs such as inappetence, diarrhoea, vomiting and jaundice within three days with history of post-exposure to rats. Another four dogs died (titre of 1:200), in which serovars Australis (n = 2) and Icterohaemorrhagiae (n = 2) were detected. The highest MAT serological titre obtained were at 1:800 (n = 8) that could suggest a severe condition in these infected dogs. Only two dogs died with a titre of 1:800 (Javanica), whilst six other infected dogs survived post-treatment (2-Javanica; 1-Hardjobovis; 1-Pomona; 1-Copenhageni; 1-Malaysia, respectively). This shows that different level of titre detected does not correlate with the risk of mortality and prognosis. On the other hand, six dogs with high infection titres did survive and therefore perhaps the dogs’ immunity level, infecting serovar and treatment initiated might all have played important roles in the survivability of infected dogs.
The panel of leptospiral serovars selected in this study was based on the important serovars circulating in Malaysia for human, rats and dogs as found in the previous studies [19, 20, 21, 22, 23, 27]. It was observed that the three most frequent leptospiral serovars detected in diseased dogs were Bataviae (n = 12), Javanica (n = 10) and Icterohaemorrhagiae (n = 10), then followed by Ballum (n = 3), Australis (n = 3), Hardjobovis (n = 3), Malaysia (n = 3) and Pomona (n = 2). The least frequent leptospiral serovars observed in this study were Canicola, Lai, Pyrogenes, Copenhageni, Celledoni, Cynopteri and Autumnalis. Therefore, incorporating known local leptospiral serovars is highly recommended in the diagnostic workout to improve detection rate as these serovars had shown to cause disease among dogs. Locally, both serovars Bataviae and Javanica had been reported in dogs [19, 21] and high serological detection of serovars Bataviae and Javanica (32 out of 53 seropositive dogs) was directly linked to direct contact with rats. This is not surprising as Benacer et al., states that Bataviae and Javanica are two leptospiral serovars circulating among the urban rats’ population in Peninsular Malaysia . Besides that, Icterohaemorrhagiae, Australis, Pomona and Canicola had been detected in rats within Kuala Lumpur  which further supports the findings in the current study. In contrast, serovars Hardjobovis and Copenhageni were commonly reported in working dogs from livestock farms in New Zealand . Thus, the dogs there were at greater risk of exposure to those serovars and detection was governed by local endemicity which was similarly observed in this study.
Direct PCR on specimens enables rapid and direct diagnosis, in both early and convalescent stages of infection. In this study, the overall molecular detection of leptospiral infection in dogs diagnosed with kidney and/or liver disease was 42.7%. Results were consistent with study by Miotto et al., at 42.4% (14/33; 95%CI: 25.6% − 59.3%) . Despite having a smaller sample size in that study, the target population was similar. In contrast, Santanna et al., and Latosinski et al., reported a lower molecular detection rate at 19.8% (26/131; 95%CI: 13.0% − 26.7%) and 1.0% (1/106; 95%CI: 0.0% − 2.8%), respectively [32.33]. Despite the similarity in large sample size with this study, the target population recruited were apparently healthy dogs which could explain the lower detection rate. The PCR can detect leptospiral DNA in the whole blood, down to extremely small amounts equivalent to the DNA content of about 10 leptospires or less . In this study, 42 pathogenic Leptospira spp. (33.9%; 42/124; 95%CI: 25.5% − 42.2%) were detected in the whole blood and suggestive of leptospiraemia phase. Out of 42 dogs diagnosed with leptospirosis, 21 dogs were presented at the acute stage as Leptospira spp. with positive detection only from the blood samples. Sixteen dogs were positive for both PCR and MAT, which suggested that the dogs were in convalescent phase, where antibodies might have started to react with the antigens, and that could reduce the amount of leptospires circulating in the body, but still detectable using the molecular method.
The optimum time of Leptospira spp. evident in the urine of infected dogs were reported at seven or more days of clinical illness [26, 34]. Positive detection of 36 pathogenic Leptospira sp. from urine samples suggested a leptospiruria phase in these diseased dogs. Twenty-three out of 36 dogs were presented at the convalescent phase. Twenty-five dogs detected positive for pathogenic Leptospira sp. in both whole blood and urine samples and 14 of the diseased dogs were presented with chronic stage, either in the period of active infection and/or actively shedding. Using the conventional PCR method, blood samples allowed higher detection compared to urine samples. This could be related to samples obtained at different phases of infection or the biological feature of urine itself. There are limitations to PCR with regards to urine samples as urea (act as PCR inhibitor) may lead to polymerase degradation affecting the sensitivity of the assay or even leads to false-negative results , which is a challenging issue with direct detection from the clinical sample obtained.
Only two kidneys and two livers were positive for pathogenic Leptospira spp. Low detection from tissue samples could be due to the presence of PCR inhibitors such as haemoglobin and hormones . However, to our knowledge, this study could be the first to demonstrate all four abdominal effusion samples obtained from four different dogs were tested positive for pathogenic Leptospira spp. and perhaps abdominal effusion can be the preferred sample for molecular diagnostic investigation. Cerebrospinal fluid (CSF) was not collected in this study, but leptospiral DNA has been expressed in CSF fluids from both, human [36, 37] and animal  studies. None of the dogs in this study showed neurological signs,
The partial 16S rRNA sequencing performed after direct PCR revealed that the most common species detected were L. interrogans (n = 62) followed by L. borgpetersenii (n = 17), L. kirschneri (n = 6), and L. kmetyi (n-1). Leptospira interrogans were detected in all type of samples (blood, urine, abdominal effusion, kidney, and liver). Comparatively similar, L. kirshneri was detected in all sample types except in liver. The detection of L. interrogans and L. kirshneri were expected in this study and both species had been commonly associated with canine leptospirosis [1, 31, 39, 40]. However, the detection of L. borgpetersenii was linked to contacts with rats as this bacterium is commonly shed by rats [28, 41, 42]. In this study, nine out of 17 samples from a total of six dogs had history of in contact with rats. Two out of the six dogs did not survive despite aggressive treatment therapy. Previous study in Germany reported three out of 200 healthy dogs shed leptospires of the species L. interrogans (n = 2) and L. borgpetersenii (n = 1) . Even though L. borgpetersenii is not common in dogs but this species remains a contributing concern to canine leptospirosis with high mortality. Leptospira kmetyi was detected from a dog’s blood sample and to our knowledge, could be the first report of L. kmetyi identified in an animal. This could be associated with environmental exposure because L. kmetyi had been isolated from the environment in Malaysia [43, 44].
Isolation and identification of leptospires are critical to confirm the specific leptospiral serovar circulating in this group of dogs diagnosed with kidney and/or liver disease. However, culturing leptospires is challenging due to frequent contamination and the fastidious growth of the pathogen . Leptospires are slow-growing bacteria in comparison to other bacteria. Even though the semisolid EMJH is the selective medium containing 5-FU to improve leptospiral isolation, however high number of contaminants greater leptospires in the samples may likely suppress the growth of the selected bacteria. Despite the 12 weeks challenges of continuous checking and sub-culturing, contaminated samples may alter the possibility of a result. Besides that, recovering leptospires from suspected dogs were limited due to early antibiotic therapy intervention, which is usually required after the disease is suspected . Nonetheless, culturing leptospires still stands as the gold standard reference test for confirmation of leptospiral infection, and only serological characterisation of the isolated strains may provide reliable information regarding serovar identity .
Despite the challenges faced throughout the study, 11 Leptospira sp. isolates were recovered from whole blood (n = 3), urine (n = 7) and abdominal effusion (n = 1) samples. Comparing with previous studies, leptospires were successfully recovered only from urine sample of the diseased dogs , shelter and stray dogs  and farm dogs . This supports that urine samples are superior samples for leptospiral isolation in dogs regardless of the target population. Leptospires can persist in the kidneys and colonize the renal proximal tubules, causing live bacteria excretion in the urine . Leptospiruric dogs remain as potential shedders and therefore, may increase the risk of infection to their owners or from dog-to-dog within the same household. The previous study found that prolonged dog handler-dog contact time increases the risk of seropositivity, and the unknowing handling of infected dogs puts the dog handlers at risk from the leptospires shedding . Sometimes, persistence leptospiruria due to inadequate antibiotic usage of failing to penetrate kidneys may ineffectively eradicate leptospires . Therefore, the use of a sensitive molecular technique is recommended to investigate as to whether an infected treated dog is still leptospiruric and/or the post-antibiotics therapy is effective. Besides urine, leptospires were commonly isolated from whole blood in human [50, 51, 52]. According to Gompf, other body fluids (besides blood and urine) might contain leptospires, but the opportunity to isolate them is slim . Surprisingly, this study successfully cultured Leptospira sp. from abdominal effusion.
Further serological characterisation of the 11 isolates showed reaction towards three hyperimmune sera namely: Bataviae (n = 8), Javanica (n = 1) and Australis (n = 2). The molecular characterisation revealed that all isolates were pathogenic and further confirmed species of L. interrogans (n = 10) and L. borgpetersenii (n = 1) by partial 16S rRNA sequencing. These results strengthen the serological and molecular detection as discussed earlier. Phylogenetic tree analysis revealed that all isolates were within pathogenic clade as follows; eight isolates closely related to reference isolate of L. interrogans serovar Bataviae, two isolates closely related to reference isolate of L. interrogans serovar Australis, and one isolate closely related to reference isolate of L. borgpetersenii serovar Javanica. Phylogenetic analysis concurred with the findings of previous reports that the pathogenic, intermediate, and saprophytic species each formed one clade [54, 55].
The isolation of serovars Bataviae, Javanica and Australis could be alarming due to their absence in commercial vaccines. In Malaysia, bivalent (Canicola and Icterohaemorrhagiae) and tetravalent vaccines (Canicola, Icterohaemorrhagiae, Pomona and Grippotyphosa) were adopted based on the availability of imported vaccine and WSAVA guidelines . Referring to the history obtained from pet owners, three diseased dogs were vaccinated with tetravalent vaccine and five were not vaccinated. Dogs vaccinated annually remained at risk as vaccination does not provide cross-protection towards non-vaccinal serovars. In Australia, commercial vaccines for dogs containing serovars of Icterohaemorrhagiae and Australis have been marketed for use . Meanwhile, trivalent (Canicola, Icterohaemorrhagiae, and Grippotyphosa) and tetravalent vaccines (Canicola, Icterohaemorrhagiae, Grippotyphosa, and Australis) have been licensed in European countries. The decision of in-cooperation incorporating serovars for optimal protection was made due to the observed shift in serovar prevalence in Europe and Australia with emerging serovar Australis .