Abstract: Background and Objectives: Leptospirosis is an important cause of reproductive losses in dairy cows. This study aimed to describe the history, clinical presentation, hematological and serum biochemical changes in Leptospira serovars Hardjo and Pomona seropositive dairy cows with recent history of abortion. Materials and Methods: A total of 40 aborted and 20 apparently healthy (control) cows were used in the study. In all aborted cows, abortion had occurred within 14 days prior to the study while control cows had no history of abortion within the previous 12 months. All cows were subjected to a complete physical examination including rectal temperature, heart rate, respiration rate, mucous membrane color, capillary refill time and rumen motility. Whole blood samples were collected from all cows for hematology and serum biochemical analysis using routine laboratory methods. The seropositivity of the cows against Leptospira serovars Hardjo and Pomona was determined using IgM-specific ELISA kits according to the manufacturers recommendations. According to the ELISA test results, cows were divided into 3 groups; Group 1 included cows seropositive to Leptospira serovar Hardjo (n = 20), Group 2 included cows seropositive to Leptospira serovar Pomona (n = 20) and Group 3 (n = 20) included control cows (non-aborted and Leptospira seronegative). Results: None of the farmers of the aborted cows have reported to notice any abnormal clinical signs before, during or after the incident of abortion. There were no statistically significant differences in any of the physical examination parameters between the 3 groups. Leptospira seropositive cows had statistically significant (p<0.05) decrease in RBC, Hb and PCV and a significant (p<0.05) increase in MCV values compared to control cows. In seropositive cows, there were statistically significant (p<0.05) increase in AST, ALP and fibrinogen values compared to control cows. No other significant differences were observed in any of the parameters in the study. Conclusion: Results of this study provide some indicators of the general health status of aborted dairy cows with previous exposure to Leptospira serovars hardjo and pomona.
INTRODUCTION
Leptospirosis is a multisystemic disease caused by the bacteria Leptospira of the Spirochetes family1-4. Leptospirosis affects a wide range of hosts including ruminants, dogs, horses, rodents, wild animals and humans1. The disease is associated with significant economic impact due to great losses in animal production, reproductive failure, treatment and prevention costs5. The disease is considered also a significant threat to public health especially to farmers, veterinarians and communities vulnerable to flooding6,7. In cattle, Leptospira serovar Hardjo is considered the most prevalent cause of abortions in the United States and Canada8.
Leptospira serovars Hardjo and Pomona are associated with multisystemic clinical effects in cattle9. In the acute form, the disease is generally characterized by no or relatively mild clinical signs including fever, icterus, hemoglobinuria and high mortality in calves10. Reproductive failure in adult cows is usually significant and characterized by widespread abortions, retained placenta, stillbirths and birth of weak calves11,12. In Jordan, recent estimates have suggested that approximately 50% of recently aborted cows and 25% of apparently healthy cows have been exposed to one or more serotype of Leptospira spp.2 These data indicates that leptospirosis is a major cause of abortion and reproductive losses in Jordan and that further research is warranted to understand the epidemiology, spread and health effects of the disease in cattle.
The clinico-pathological studies of infectious diseases provide important clues that reflect the systemic impact and severity of the disease on various body organs which may be used for disease diagnosis and case follow-up after initiation of appropriate treatment. The clinical, hematological and serum biochemical effects of leptospirosis have been reported in many animal species including sheep, goats, horse, rats and dogs13-16. However, in dairy cows such information is currently lacking. Therefore, in this study the objectives were to study the clinical, hematological and serum biochemical effects of leptospirosis in recently aborted cows.
MATERIALS AND METHODS
Animals: All procedures performed in this study were approved by the Jordan University of Science and Technology Animal Use and Care Committee. The study was carried out during the summer of 2018 between the months of June to September using 40 aborted and 20 apparently healthy cows. Aborted cows were presented to field veterinarians in private farms located in Irbid and Al-Dulail regions of Jordan for health and reproductive evaluations after confirmed incident of abortion.
Selection criteria: To be included in the study, cows must have undergone an abortion within the previous 21 days with no other apparent disease conditions. For control, cows must be completely healthy on physical examination and must have no history of abortion within the last 12 months prior to the study.
Clinical examination: All cows were subjected to complete physical examination to determine their health status including rectal temperature, heart rate, respiration rate, mucous membrane color, capillary refill time and rumen motility. For control, 20 apparently healthy cows were randomly chosen from the same farms were aborted cows came from. Physical examination in aborted and control cows was performed by the principle investigator or by trained resident farm veterinarians.
Blood sample collection: Approximately 10 mL of whole blood was collected from aborted and control cows via jugular venipuncture using hypodermic needle and vacutainer blood tubes. Blood samples were placed in plain and in EDTA-containing blood tubes and transferred to the laboratory on ice within 4-6 h. Serum was then obtained by centrifugation of clotted samples at 3000 g for 10 min. Obtained serum samples were stored at -20°C until laboratory analysis was performed.
Leptospira serovars hardjo and pomona serology: To determine the seropositivity of aborted and control cows, a quantitative sandwich IgM-specific ELISA kit against Leptospira serovars hardjo and pomona was used according to manufacturer’s recommendations (Sincere Bioteck, China). Positive and negative sera controls were provided with the ELISA kit and 3 replicates of each control were used per plate. The minimum detection limit of IgM of this ELISA kit was 61.5 pg mL–1. This minimum limit was used to classify the results as positive and negative. The ELISA plate was read at 450 nm wavelength using a microplate reader (Thomas Scientific, USA).
Hematology analysis: In the hematology analysis, the following parameters were determined using automated hematology analyzer (Scil Vet ABC Hematology Analyzer, Scil Animal Care Company, USA): Red blood cell count (RBC), packed cell volume (PCV), hemoglobin concentration (Hb), platelets count, mean corpuscular volume (MCV), mean corpuscular hemoglobin concentration (MCHC) and white blood cell count (WBC). The differential leukocyte count including the percentages of neutrophils, lymphocytes, monocytes, basophils, eosinophils were determined manually using blood smears stained with Giemsa stain.
Serum biochemistry analysis: For the serum biochemical analysis, the following parameters were determined using commercially available kits and reagents (Randox Laboratories, UK) according to manufacturer’s instructions17. The following parameters were determined: glucose, total protein (TP), albumin, blood urea nitrogen (BUN), creatinine, aspartate transaminase (AST), alanine transferase (ALT), alkaline phosphatase (ALP). Serum concentrations of calcium, potassium, magnesium, phosphorus, sodium and chloride were determined using automated analyzer (Awareness Technology, Inc., USA).
Statistical analysis: To perform the statistical analysis, cows were divided according to the results of the ELISA test into 3 groups: Group 1 (n = 20) includes cows that were seropositive against Leptospira serovar Hardjo, Group 2 (n = 20) includes cows that were seropositive against Leptospira serovar pomona and Group 3 (control; n = 20) includes apparently healthy and seronegative cows against both serovars. Data were expressed in Means±SD. Independent t-test was used to compare various clinical, hematology and serum biochemistry parameters between seropositive and control cows. Statistical analysis was performed using SPSS software (SPSS Inc., Chicago, Illinois). Values of p<0.05 were considered significant.
RESULTS
Historical findings: Abortion occurred in cows within the last 2 weeks prior to the study in all cows in groups 1 and 2. Cows in group 3 had no history of abortion within the last 12 months prior to the study. None of the farmers of the aborted cows have reported to notice any abnormal clinical signs before, during or after the incident of abortion.
Clinical and epidemiological findings: The clinical and epidemiological data of cows used in this study are presented in Table 1. The mean±SD of the cow's age was 3.5±1.0, 3.9±1.0 and 2.7±0.5 years in group 1, group 2 and group 3, respectively. The average body condition score and days-in-milk of cows involved in the study were similar in all groups. There were no statistically significant differences in any of the physical examination parameters between the 3 groups. Physical examination revealed that the heart rate, respiratory rate, rectal temperature, rumen motility, mucous membrane color and capillary refill time were all within normal limits.
Hematology analysis findings: The hematology parameters in Leptospira seropositive cows with recent history of abortion are presented in Table 2. Compared to apparently healthy cows, Leptospira seropositive cows had statistically significant (p<0.05) decrease in RBC, hemoglobin and packed cell volume while values of mean cell volume were significantly (p<0.05) increased. The values of total white cell count and the percentages of neutrophils and lymphocytes were increased but not significantly compared to their values in control cows.
| Table 1: | Clinical and epidemiological data of Leptospira serovars hardjo and pomona seropositive dairy cows with recent history of abortion (Mean±SD) |
| Table 2: | Hematology parameters of Leptospira serovars hardjo and pomona seropositive dairy cows with recent history of abortion (Mean±SD) |
*p<0.05 | |
| Table 3: | Serum biochemistry analysis of Leptospira serovars hardjo and pomona seropositive dairy cows with recent history of abortion (Mean±SD) |
*p<0.05 | |
Serum biochemistry analysis: The serum biochemical values in Leptospira seropositive cows with recent history of abortion are presented in Table 3. There were statistically significant (p<0.05) increase in serum AST and ALP activities while values of ALT were non-significantly increased in seropositive cows. The fibrinogen concentrations in seropositive cows were significantly (p<0.05) increased. There was a non-significant increase in blood urea nitrogen values in seropositive cows compared to control cows.
DISCUSSION
Leptospirosis is an important cause of reproductive loss in dairy cattle1. The most important Leptospira serovars affecting dairy cattle are hardjo and pomona1. In this study, the history, clinical presentation, hematology and serum biochemical changes in Leptospira serovars hardjo and pomona seropositive dairy cows with recent history of abortion were determined for the first time. In accordance with previous studies regarding bovine abortion, there were no significant alterations in any of the clinical examination parameters in seropositive cows3. Cattle are considered the maintenance host of Leptospira serovar hardjo1. Infection of the maintenance host by Leptpsira spp. is usually chronic in nurture and clinically characterized by reproductive manifestations such as abortions, birth of weak calves, stillbirths and premature birth1. It has been reported that serological antibody titers in case of maintenance serovars are high at the time of abortion4. This fact coincides well with the serological diagnosis in this study where IgM-specific ELISA test was used to classify exposure to Leptospira serovars hardjo and pomona. While the microscopic agglutination test (MAT) is considered the gold standard diagnostic test for leptospirosis, recent studies have indicated that IgM-specific ELISA has similar sensitivity and specificity as MAT and may be superior to MAT during the acute phase of the disease and as such is more suitable for early diagnosis of leptospirosis18. This could provide a circumstantial evidence that the abortion reported here in this study is in fact due to Leptospira infection.
In this study, Leptospira seropositive cows had statistically significant decrease in RBC, Hb and PCV and a significant increase in MCV values compared to control cows. These results are similar to those reported in Leptospira seropositive goats16, dogs15 and Wistar rats14. In horses however, only non-significant alterations in some hematological parameters were detected in Leptospira serovar Bratislava seropositive horses13. In general, host adapted Leptospira serovars are known to cause mild or no clinical signs including hematological alterations besides the reproductive effects13. In the serum biochemical analysis, seropositive cows showed statistically significant increase in AST, ALP and fibrinogen values compared to the control cows. These results are partially in congruence with results obtained in seropositive goats where a significant increase was reported in serum ALT, AST, BUN, creatinine, cholesterol and total bilirubin concentrations while levels of TP were significantly decreased16,19. In cows, the levels of total bilirubin, ALT and AST were found increased in seropositive cows20. In dogs, the serum levels of ALP, ALT, urea and creatinine were significantly increased15,21. In horses, the ALP, ALT, total bilirubin, direct bilirubin and creatinine were significantly increased13. In Wistar rats, the ALP, ALT, urea and creatinine were increased14. However, no abnormal changes were detected in sheep affected with leptospirosis22.
Although, previous research have suggested significant hepatic and renal damage in animals affected with leptospirosis23, none of the biochemical parameters related to renal function were significantly altered indicating no significant renal damage has occurred. This could be because normal renal function continues until the majority of kidney tissues are affected.
CONCLUSION
In conclusion, results of this study provide some indicators of the general health status of dairy cows with recent history of abortion and recent exposure to Leptospira serovars hardjo and pomona.
SIGNIFICANCE STATEMENT
Leptospirosis is an important zoonotic disease that may result in significant production losses in dairy cows. Most natural infections in adult dairy cows are asymptomatic and therefore, knowledge of possible changes within the hematological and serum biochemical parameters become an important tool to evaluate the general health status of affected herds. Results of this study provide some indicators of the general health status of dairy cows with recent history of abortion and recent exposure to Leptospira serovars hardjo and pomona.
ACKNOWLEDGMENT
Thanks to the Deanship of Research at Jordan University of Science and Technology for their financial support (Grant Number 325/2019).