Volume 16, Issue 4 (12-2025)
Res Anim Prod 2025, 16(4): 64-73 |
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Tahouri L, Moravedji M, Abdollahpour G, Sadeghi Alavije J. (2025). Serological and Etiological Investigation for Diagnosing Bovine Leptospirosis in Markazi Province. Res Anim Prod. 16(4), 64-73. doi:10.61882/rap.2025.1540
URL: http://rap.sanru.ac.ir/article-1-1540-en.html
URL: http://rap.sanru.ac.ir/article-1-1540-en.html
1- Department of Clinical Sciences, Sa.C., Islamic Azad University, Sanandaj, Iran
2- Department of Internal Medicine, Faculty of Veterinary Medicine, University of Tehran, Tehran, Iran
3- Department of Clinical Pathology, Faculty of Veterinary Medicine, University of Tehran, Tehran, Iran
2- Department of Internal Medicine, Faculty of Veterinary Medicine, University of Tehran, Tehran, Iran
3- Department of Clinical Pathology, Faculty of Veterinary Medicine, University of Tehran, Tehran, Iran
Abstract: (1180 Views)
Extended Abstract
Background: Leptospirosis, caused by serovars of the bacterium Leptospira interrogans, is one of the most dangerous zoonotic diseases. It produces a wide range of sub-acute to acute clinical signs and can lead to infertility, abortion, reduced production, and significant economic losses to Iran's livestock industry. Leptospirosis diagnosis is of particular importance due to its potential risks to human communities, as the disease is easily transmitted by hosts such as rats and dogs, which are often found in significant numbers in livestock environments. However, since this disease can cause highly misleading symptoms in animals, such as mucosal jaundice, swollen lymph nodes, uveitis, hematuria, mastitis, a sharp drop in milk production and discoloration, abortion, birth of weak calves, weight loss, and fertility disorders, it increases the likelihood of diagnostic errors by veterinarians, thereby affecting the health status of human communities. Various laboratory methods can be used to diagnose the disease, including direct microscopic observation, culture, PCR, the Microscopic Agglutination Test (MAT), Enzyme-Linked Immunosorbent Assay (ELISA), and Immunofluorescence Assay (IFA). This research aimed to study and compare three methods—serology, culture, and PCR—for diagnosing bovine leptospirosis.
Methods: In this study, blood and urine samples were collected from 400 heads of cattle with a history of at least one symptom of leptospirosis. Blood was drawn from the jugular vein under hygienic conditions. Serum samples were used for serological testing (MAT). Serum was separated from clotted blood tubes by placing them in a 37 °C water bath for 20 min, followed by centrifugation at 3000 × g for 10 min. To preserve the separated sera until the start of the MAT test, they were transferred using a sampler to sterile 2-mL microtubes and stored at -20 °C. The standard MAT procedure was as follows. The antigen used in this method (Leptospira interrogans) was cultured in a liquid medium for 7-10 days. The concentration of leptospires used was evaluated by counting in a chamber, and the volume was adjusted to contain 2 × 10^8 leptospires per milliliter. Urine samples were used for urine culture and PCR testing. Primers specific to L. interrogans were evaluated for the PCR assay. PCR reactions were set up in 25 µL volumes using a ready-made master mix (BIOFACT Company, Korea; composition: ddH2O: 5.50 µL, Buffer: 12.50 µL, Forward primer: 1.00 µL, Reverse primer: 1.00 µL, DNA: 5.00 µL). The required urine volume was 50 mL, collected in bottles containing 0.5 mL of filtered formaldehyde (0.45 µm). For bacterial culture and isolation, 5 µL of the collected urine was inoculated into a specific bacterial culture medium (semi-solid EMJH medium). Culture media were incubated at 30 °C for at least 5 weeks and examined every week for leptospiral growth using Dark-Field Microscopy (DFM). Data were analyzed using the Chi-square test and Fisher's exact test with SPSS statistical software version 23.0 (SPSS, Inc., Chicago, IL, USA, 2007). Significant differences between means were considered at a significance level of P < 0.05.
Results: The MAT test was positive in five heads of cattle (1.2%). The most common serovars were Icterohaemorrhagiae (2 head = 40%), Grippotyphosa (2 head = 40%), and Canicola (1 head = 20%). All five samples that tested positive by MAT, plus all cattle showing clinical signs of leptospirosis, were candidates for PCR testing, four heads of which (1%) yielded a positive result. Urine cultures were prepared for all suspected cattle with positive MAT, PCR, and clinical signs. Despite the contamination of two culture media with environmental bacteria, none of the other media showed contamination, and no growth of Leptospira was observed (0%). Since animals in the early stages of the disease, whose immune systems have not yet completely cleared the bacteria from the blood, may not have allowed sufficient time for bacterial colonization in the kidneys; this could lead to negative urine samples. Additionally, Leptospira bacteria are sensitive to many antibiotics (treatment is doxycycline and azithromycin); therefore, these antibiotics may be used in cases where the animal has another disease, resulting in bacterial elimination and negative cultures.
Conclusion: The results of this study indicated that the prevalence of Leptospira infection in cattle in Markazi Province was much lower than in other studies, which is why an accurate comparison of the three methods was not feasible. An interesting point in this study was the lack of overlap between positive tests in the MAT and PCR methods, suggesting that more studies are needed for a more precise comparison of these two methods. Based on the results of this research, the urine culture method is not recommended due to its lengthy process and the specific conditions required for bacterial colonization.
Background: Leptospirosis, caused by serovars of the bacterium Leptospira interrogans, is one of the most dangerous zoonotic diseases. It produces a wide range of sub-acute to acute clinical signs and can lead to infertility, abortion, reduced production, and significant economic losses to Iran's livestock industry. Leptospirosis diagnosis is of particular importance due to its potential risks to human communities, as the disease is easily transmitted by hosts such as rats and dogs, which are often found in significant numbers in livestock environments. However, since this disease can cause highly misleading symptoms in animals, such as mucosal jaundice, swollen lymph nodes, uveitis, hematuria, mastitis, a sharp drop in milk production and discoloration, abortion, birth of weak calves, weight loss, and fertility disorders, it increases the likelihood of diagnostic errors by veterinarians, thereby affecting the health status of human communities. Various laboratory methods can be used to diagnose the disease, including direct microscopic observation, culture, PCR, the Microscopic Agglutination Test (MAT), Enzyme-Linked Immunosorbent Assay (ELISA), and Immunofluorescence Assay (IFA). This research aimed to study and compare three methods—serology, culture, and PCR—for diagnosing bovine leptospirosis.
Methods: In this study, blood and urine samples were collected from 400 heads of cattle with a history of at least one symptom of leptospirosis. Blood was drawn from the jugular vein under hygienic conditions. Serum samples were used for serological testing (MAT). Serum was separated from clotted blood tubes by placing them in a 37 °C water bath for 20 min, followed by centrifugation at 3000 × g for 10 min. To preserve the separated sera until the start of the MAT test, they were transferred using a sampler to sterile 2-mL microtubes and stored at -20 °C. The standard MAT procedure was as follows. The antigen used in this method (Leptospira interrogans) was cultured in a liquid medium for 7-10 days. The concentration of leptospires used was evaluated by counting in a chamber, and the volume was adjusted to contain 2 × 10^8 leptospires per milliliter. Urine samples were used for urine culture and PCR testing. Primers specific to L. interrogans were evaluated for the PCR assay. PCR reactions were set up in 25 µL volumes using a ready-made master mix (BIOFACT Company, Korea; composition: ddH2O: 5.50 µL, Buffer: 12.50 µL, Forward primer: 1.00 µL, Reverse primer: 1.00 µL, DNA: 5.00 µL). The required urine volume was 50 mL, collected in bottles containing 0.5 mL of filtered formaldehyde (0.45 µm). For bacterial culture and isolation, 5 µL of the collected urine was inoculated into a specific bacterial culture medium (semi-solid EMJH medium). Culture media were incubated at 30 °C for at least 5 weeks and examined every week for leptospiral growth using Dark-Field Microscopy (DFM). Data were analyzed using the Chi-square test and Fisher's exact test with SPSS statistical software version 23.0 (SPSS, Inc., Chicago, IL, USA, 2007). Significant differences between means were considered at a significance level of P < 0.05.
Results: The MAT test was positive in five heads of cattle (1.2%). The most common serovars were Icterohaemorrhagiae (2 head = 40%), Grippotyphosa (2 head = 40%), and Canicola (1 head = 20%). All five samples that tested positive by MAT, plus all cattle showing clinical signs of leptospirosis, were candidates for PCR testing, four heads of which (1%) yielded a positive result. Urine cultures were prepared for all suspected cattle with positive MAT, PCR, and clinical signs. Despite the contamination of two culture media with environmental bacteria, none of the other media showed contamination, and no growth of Leptospira was observed (0%). Since animals in the early stages of the disease, whose immune systems have not yet completely cleared the bacteria from the blood, may not have allowed sufficient time for bacterial colonization in the kidneys; this could lead to negative urine samples. Additionally, Leptospira bacteria are sensitive to many antibiotics (treatment is doxycycline and azithromycin); therefore, these antibiotics may be used in cases where the animal has another disease, resulting in bacterial elimination and negative cultures.
Conclusion: The results of this study indicated that the prevalence of Leptospira infection in cattle in Markazi Province was much lower than in other studies, which is why an accurate comparison of the three methods was not feasible. An interesting point in this study was the lack of overlap between positive tests in the MAT and PCR methods, suggesting that more studies are needed for a more precise comparison of these two methods. Based on the results of this research, the urine culture method is not recommended due to its lengthy process and the specific conditions required for bacterial colonization.
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