*5.1.2.2 Isothermal methods*

*Bacterial Cattle Diseases*

acid is required [51].

*5.1.1 Direct examination*

*5.1.2 Gene amplification*

*5.1.2.1 PCR*

Infection occurred by pathogenic leptospires is divided into two stages, first stage is acute stage or septicaemia (because septicaemia is in this stage), which lasts from 7 to 10 days with headache and myalgia. The second stage is immune stage which is after first week of infection and lasts 4–30 days [34]. During first stage, leptospires are present in blood and can say bacterial count is high in the blood, while when second stage starts, then the level of antibodies IgM and IgG start to increase and this increase in antibodies titer is correlated to elimination of leptospires from blood. *Leptospira* antigens and DNA sometimes may not be detected from the blood; this may be due to late sampling, or sampling in acute stage where proper level of leptospiremia is not developed and due to antibiotic administration, leptospires are eliminated from the blood. False negative results will be there, if we detect antibodies prior to sero-conversion during acute stage. Coagulated blood has serum and clot while non-coagulated blood has plasma, RBCs, WBCs and platelets. That can be collected according to the tests. If you are going to do gene amplification than EDTA, plasma gives the best results [50]. Leptospires can be detected in urine and cerebrospinal fluid samples. Many kits are available in the market for rapid detection of leptospires from blood, urine, and CSF sample; these kits basically detect nucleic acid of leptospires, but for these tests, purification of nucleic

**5.1 Current tools and emerging technologies for diagnosis of** *Leptospira*

Different tools are being developed for the study of virulence factors, pathogenicity, and basic cell biology of organisms [52]. These are essential for proper treatment and reduction of the severity of the disease. During acute infection, nonspecific symptoms of leptospires mimic the febrile condition, which are essential for proper treatment and reduce the severity of the disease. Therefore, the diagnosis of leptospirosis is highly dependent on the particular laboratory tests [13]. Serology is the dominant one in diagnosis, while the micro-aggregation test (MAT) is the standard serological reference method. MAT is a sensitive test due to the antigenic heterogeneity of *Leptospira*, which require a large number of serovars as antigens. Furthermore, it is useless at early stage of the disease when antibodies are not present or present in less quantity [18]. Detection of disease in early stage helps the epidemiological investigators. However, antigen detection at this stage is more expensive and complex [13]. Current diagnostic tools for *Leptospira* detections other than MAT are rapid antibodybased tests, direct examination of blood, the rapid nucleic-acid diagnosis, [53], dark field microscopy (DFM), IgM ELISA, and polymerase chain reaction (PCR) [13].

This method is cheap, but for direct examination, dark field microscope is required

–10<sup>−</sup><sup>6</sup>

[54]. Theoretically, leptospires may be diagnosed by direct examination of blood during first week after onset of symptoms. Leptospires are 6–20 μm long and their diameter is 0.15 μm. Because of their size, dark field microscopy is required; 10<sup>−</sup><sup>2</sup>

leptospires/mL of blood may be observed during the acute stage of leptospirosis [55].

The use of PCR is increasing in recent years and it has replaced the serological methods in endemic areas, because it is more sensitive and has capacity to give early

**5. Diagnosis**

**50**

In recent years, many isothermal amplification techniques are developed like isothermal technique [59]. This technique can be used as alternative to the PCR. There is no need for constant maintenance of temperature at 60–65°C and no thermal recycler is required; so, these things make it best for developing countries.

**Figure 4.** *Sensitivity of different diagnostic tests during acute phase of leptospirosis [58].*

For this, an effective and specific amplification is performed by DNA polymerase and six primers in 1 hour under isothermal conditions. Now the amplified DNA can be easily detected by eye observation of fluorescence without using gel electrophoresis [60]. Loop-mediated isothermal amplification (LAMP) methods are recently developed for the quick diagnosis of pathogenic leptospires, and lipL41 and rrs are the genes targeted by LAMP. The specificity of these methods is weak because these can detect the threshold between 2 and 100 leptospires/reactive mixture [61].
