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Microscopic Agglutination Test (MAT) for Leptospirosis in Association with Acute Renal Failure in Dogs

The present investigation was carried out in the Department of Veterinary Medicine, College of Veterinary and Animal Sciences, GBPUA&T, Pantnagar, Uttarakhand, India. Total of 100 sera samples from azotemic dogs were subjected to Microscopic Agglutination Test (MAT) using Leptospira culture in Leptospira lab, Department of Veterinary Bacteriology and Mycology, IVRI, Izatnagar, Bareilly (U.P.) All the sera samples were tested against L. interrogans: Serovars icterohaemorrhagiae, pomona, canicola, autumnalis, grippotyphosa and javanica. Out of 100 sera samples, 20 (20%) were found positive (+) for different serovars of Leptospira interogans and 3 (3%) were doubtful (±) at an antibody titre ≥1:100. The serovars identified were L. autumnalis (17/100), L. icterohaemorrhagiae (8/100), L. gryppotyphosa (4/100), L. canicola (2/100), L. javanica (2/100) and L. Pomona (1/100). The highest prevalence of Leptospira was observed for serovars L. autumnalis (17%), followed by L. icterohaemorrhagiae (8%) and least for L. pomona (1%).

Renal failure is a common clinical manifestation in 2-5% of dogs and third leading cause of deaths in canine (Bronson, 1982;Lund et al., 1999). The mean age of diagnosis is 6.5 years with 45% of cases reported over 10 years of age (Srivastava et al., 2011). About 30.77% acute kidney injuries in dogs caused by leptospirosis (Segev et al., 2008). Acute renal failure (ARF) is a multi-systemic disease involving usually more than one organ and causes rapid decline in renal function, resulting in acute uraemia, fluid retention, acid-base and electrolyte imbalances (Francey and Cowgill, 2002;Patias, 2002).
Dogs become infected by exposure to contaminated urine from shedding wild animals, mainly by rodents that act as a maintenance host for several Leptospira serovars. The Leptospira organisms penetrate the mucus membranes, reached to the liver and kidneys and damage these organs (Goldstein, 2010). The most common clinical signs observed in dogs consisted of icterus and haemorrhagic diathesis, but acute renal failure is the most prevalent clinical signs attributed now a days.
Microscopic agglutination test (MAT) and polymerase chain reaction (PCR) is most commonly used diagnostic techniques for leptospirosis (Goldstein, 2010). However, detection of Leptospira antibodies using the MAT has been the most common (Greene et al., 2006). Keeping in view the present investigation was carried out at College of Veterinary and Animal Sciences, GBPUA&T, Pantnagar, Uttarakhand, India with the objective of serological investigation of leptospiral acute renal failure in dogs.

MATERIALS AND METHODS
In the present investigation sera samples from 100 azotemic dogs belonging to different categories like vaccinated and unvaccinated, were subjected to Microscopic Agglutination Test (MAT) using Leptospira culture in Leptospira lab, Department of Veterinary Bacteriology and Mycology, IVRI, Izatnagar, Bareilly (U.P.). The results obtained are expressed in Table 1 and Fig 1. All the sera samples were tested against six serovars of Leptospira spp., most likely to cause disease in dogs in this region which are L. interrogans: Serovars icterohaemorrhagiae, pomona, canicola, autumnalis, grippotyphosa and javanica. The MAT was performed according to the method of Faine (1982). A homologous, high titred antiserum was included in each testing session. Serum dilutions were prepared in 8-well "U" bottomed disposable micro-liter plates. A serial two-fold dilution of each serum was made with phosphate buffered saline (pH 7.2) starting with an initial dilution of 1:100. An equal volume (i.e. 10 µl) of culture was added to each well, mixed by gentle rocking, and incubated at 37°C for 3 hours after sealing with polyethylene sheet. The MAT titer was the reciprocal of the highest dilution of the serum in which >50% of the antigen was agglutinated. A minimum titer of 1:100 and above was taken as the positive agglutination reaction for leptospirosis (Cole, 1973;O'Keefe et al., 2002).
Leptospirosis is a significant cause of acute tubulointerstitial nephritis before the widespread use of antileptospiral bacterins (Robertson, 1986).
The prevalence of canine leptospirosis has increased in recent years but prevalence varies by region. Results of one study in Michigan indicated that >20% of healthy, client-owned dogs were exposed to Leptospira serovars (Stokes et al., 2007).  In another study, 8.2% of dogs were shedding pathogenic leptospires irrespective of the health status (Harkin et al., 2003). Acute kidney disease is the most commonly recognized disease in dogs, accounting for more than 90% of reported cases of leptospirosis (Levy et al., 1996). The present findings are in accordance with the findings of many authors (Miller et al., 2008;Senthil et al., 2013).
It was concluded that leptospirosis is the most common cause of acute renal failure in canines resulting in death. MAT was highly reliable and rapid diagnostic method of leptospirosis in canine. It is therefore, suggested that regular serological testing (MAT) for leptospirosis in canine cane save the life of valuable dogs by early detecting the disease and proper medical care.

CONCLUSION
It is concluded that leptospirosis is a serious problems in dogs particularly in young animals and most commonly occurs in wet and humid climate. It causes serious acute renal failure (ARF) and resulted into multi-systemic organ failure. Microscopic agglutination test (MAT) is a reliable and highly sensitive serological test for leptospirosis in dogs. Vaccinated animals may show positive reaction against vaccinated serovars. So, regular MAT testing of dogs against this disease is must to save the life of valuable dogs even in early stage and also to prevent infection to the dog owner as the disease has zoonotic importance.