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A retrospective study of canine leptospirosis with emphasis to diagnosis and dark field microscopy.


Leptospirosis is a sporadic emerging zoonotic disease in Asia and South America caused by motile spirochetal bacterium of genus Leptospira (W.H.O., 1999) and widespread in tropical countries owing to their longer survivability in warm and humid environment (Zavitsanou and Babatsikou, 2008). They are classified into two species viz. Leptospira interrogans and Leptospira kirschneri (Goldstein, 2010). Several serovars of Leptospira spp. are pathogenic to both humans and animals, namely icterrohae-morrhagica, canicola, pomona and grippotyphosa.

Transmission of infection is mainly by contaminated food, water, soil and habitats of animals by urine of infected animals. Outbreaks of disease are associated with occupational exposure, tourism, sports and inadequate sanitation measures (Dzyban, 2007). Primary hosts of infection are rats, mice, skunks and moles (Goldstein, 2010). In dogs, liver and kidneys are the target organs causing fever, anorexia, vomiting, polyuria, polydypsia, haemetemesis, melena, epistaxis, renal and hepatic insufficiency, pulmonary manifestations and reproductive failures. Exposure to infection for 2-3 weeks results in chronic active hepatitis or chronic renal disease.

Diagnosis of infection is by means of Dark Field Microscopy, MAT, PCR and ultrasonography.

Materials and Methods

The study was carried out during the period from February-October' 2013 in three hundred and eighty dogs presented and screened for presence of leptospires.

Dark field microscopical examination was conducted in blood plasma of 380 dogs and urine from 23 dogs. About 5ml of blood was collected in sterile EDTA vial and urine was collected in sterile containers having phosphate buffered saline. The samples were run at 3000 rpm for 5 minutes and supernatant was checked under DFM. If no organisms were detected, the supernatant was run at 14,000 rpm for 10 minutes and pellet was examined by DFM (Krishna et al., 2012). Leptospiral organisms were detected in plasma of 149 animals and urine of 8 animals. Out of these, 30 DFM positive animals were showing clinical symptoms of fever, icterous, anorexia, vomiting, melena and haematuria for which detailed haemato-biochemical examination was conducted. Among the 30 clinically positive cases, 26 animals had undergone regular vaccination.

Results and Discussion

Out of the 380 animals screened, 157 were found to be positive in DFM plasma and urine (Table 1 and Fig. 1), of which 30 DFM positive animals showed clinical symptoms of leptospirosis such as fever, anorexia, icterous, vomiting and melena (Table 2). Prognosis was considered grave in nine animals that were clinically positive for leptospirosis (Table 3). Anorexia, vomiting and pyrexia were observed to be the most predominant clinical signs in animals diagnosed with leptospirosis. Greene (2012) also reported clinical symptoms observed in the study along with rapid dehydration, peripheral vascular collapse, tachypnoea, irregular pulse, haematochezia, haematemesis, epistaxis, widespread petechiae, hepatic and renal failure and subsequently death.


Haemato-biochemical examination revealed leucocytosis with shift to left, anaemia, low haemoglobin content, erythrocytopenia, thrombocytopenia and elevated levels of creatinine and blood urea nitrogen in clinical DFM positive animals (Table 4) with majority of animals having neutrophilia, anaemia and thrombocytopenia. These results were in accordance to clinical laboratory findings by Goldstein (2010) and Greene (2012) that included leucocytosis with a left shift, thrombocytopenia, non-regenerative anaemia, hypoalbuminemia, hyponatremia, hyperglobulinemia, elevated levels of bilirubin and alanine amino transferase, hypoglycaemia with severe hepatic failure.

All DFM positive animals were vaccinated except four. Zavitsanou and Babatsikou (2008) also observed dogs vaccinated against leptospira shedding organisms in urine thereby resulting in domestic transmission in humans. The appropriate increases in serum antibodies in host clears spirochetes from most of organs but organisms may persist in kidneys and organisms shed in urine from days to months (Greene, 2012).

Animals showing clinical signs of leptospirosis were treated with Inj. Benzylpenicillin BID administered intravenously along with other supportive and fluid therapy for one week. However, only animals in early stage of infection responded well to medication. A mortality rate of 26.67 percent was observed in animals that showed clinical signs among which seven out of eight animals that succumbed to death had elevated levels of creatinine and BUN (Table 4). The treatment adopted was in accordance with Greene (2012) who suggested administration of Benzyl Pencillin, Amoxicillin, Am picillin, Erythromycin and Doxycyline along with aggressive fluid and supportive therapy in animals affected with leptospirosis.

DFM positive animals that were clinically negative were given prophylactic treatment using Doxycycline PO SID at dose rate of 5mg/kg b. wt. for two weeks. The blood plasma of all animals was sent for dark field microscopic examination after treatment. The DFM samples were found to be negative for leptospirosis (Table 3) indicating that Doxycycline was effective against leptospires as a prophylactic drug (Kahn and Line, 2010).

Dark Field Microscopical examination aided the rapid diagnosis of animals having symptoms of leptospirosis for better surveillance (Zavitsanou and Babatsikou, 2008). This study showcases the importance of DFM in early diagnosis of leptospirosis and also urgent need for development of a modified vaccine against leptospirosis. Animals presented with clinical signs of pyrexia, anorexia and vomiting should be suspected for leptspirosis. In future, more studies should be conducted on production of vaccines which are genus specific for complete eradication of the disease. Hence, Dark Field Microscopy aids in speedy and better diagnosis of diseases and critically helps in better surveillance of animals (Krishna et al., 2012) despite certain shortcomings like lack of sensitivity and specificity (Ahmad et al., 2005).


Ahmad, S. N., Shah, S. and Ahmad, F. M. H. (2005). Laboratory diagnosis of leptospirosis. J. Postgrad. Med. 51: 195-200.

Dzyban, L.A. (2007). Leptospirosis in the North West: An Update. Tech. Bultn. 1.

Goldstein, R. E. (2010). Canine Leptospirosis. Vet. Clin. Small Anim. 40: 1091-01.

Greene, C. E. (2012). Infectious Diseases of the Dog and Cat. 4th edn., Elsevier Publications, p. 431-47.

Kahn, C. M. and Line, S. (2010). The Merck Veterinary Manual, 10th edn. Merck & Co. Inc., p. 590-96.

Krishna, V. S., Joseph, S., Ambily, R. and Mini, M. (2012). Evauation of dark field microscopy, isolation and microscopic agglutination test for the diagnosis of canine leptospirosis. Int. J. Pharm. Bio. Sci. 2: 85-89.

Zavitsanou, A. and Babatsikou, F. (2008). LeptospirosisEpidemiology and preventive measures. H S J. 2: 75-82.

(1.) Corresponding author.


A.A. Aipe (1), V.S. Jaikumar, M. Soman and U.N. Pillai

Department of Clinical Veterinary Medicine

College of Veterinary and Animal Sciences

Kerala Veterinary and Animal Sciences University (KVASU)


Thrissur--680651 (Kerala)
Table 1: Number of DFM positive animals

Number       DFM      Clinically       Un-
of         positive   leptospiral   vaccinated
animals     out of     positive       (n=30)
screened     380      out of 157

380          157          30            4
            41.32%       19.1%        13.3%

Table 2: Symptomwise categorisation of DFM positive animals with
clinical signs (n= 30)

Fever    Icterous   Anorexia   Vomiting   Melena   Haematuria

11          4          23         18        5          4
36.67%    13.33%     76.67%      60%      16.67%     13.33%

Table 3: Prognosis of DFM positive animals and response to treatment

            Prognosis of DFM Positive Animals

             Clinically   Clinically
              Negative     Positive    Grave
              (n=157)      (n=157)     (n=30)

Number          127           30         9
Percentage     80.89        19.11      30.00

                        Treatment Response

             Clinically Positive Clinically Positive
             Negative (n=127)    (n=30)
             Recovered   Death   Recovered   Death

Number          127        0        22         8
Percentage    100.00     0.00      73.33     26.67

Table 4: Haemato-biochemical categorisation of DFM positive
animals with clinical signs (n=30)

Leuco-      Neutro-   Low   Erythro-    Thrombo-     Elevated
cytosis     philia    Hb    cytopenia   cytopenia   Creatinine

8             11       9        6           9           7
26.67%      36.67%    30%      20%         30%        23.33%

Leuco-      Elevated
cytosis       BUN

8              7
26.67%       23.33%
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Title Annotation:Research Article
Author:Aipe, A.A.; Jaikumar, V.S.; Soman, M.; Pillai, U.N.
Publication:Intas Polivet
Article Type:Report
Date:Jul 1, 2015
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