Leptospirosis seroprevalence in Canadian beef calves at or near fall weaning

Abstract

Objective

Our objective was to estimate the seroprevalences of 6 Leptospira serovars in beef calves at or near fall weaning and assess how concentrations of serovar antibody titers in weaning-age calves varied with Leptospira herd vaccination programs.

Animals

Serum was collected from 1922 beef calves from 106 herds in the Canadian Cow-Calf Surveillance Network (C3SN).

Procedure

A microscopic agglutination test was used to measure antibody titers for serovars Bratislava, Canicola, Grippotyphosa, Hardjo, Icterohaemorrhagiae, and Pomona. Records of Leptospira vaccine use were available for cows and nursing calves from 94 herds. Generalized estimating equations were used to estimate the associations between serovar antibody titers in weaning-age calves and the number of doses of Leptospira vaccine administered to cows.

Results

Overall, 90.8% of sampled calves had undetectable (< 50) antibody titers to all tested serovars, whereas 3.2% of sampled calves from 26.4% of participating herds had a positive (≥ 100) titer to 1 or more serovars. The seroprevalences at both the calf and herd levels were higher for the host-adapted serovar Hardjo in western Canada and higher for the non-host-adapted serovars in eastern Canada. The probability of calf serovar Icterohaemorrhagiae titer increasing by 1 doubling dilution was 3.6× (95% CI: 1.7, 7.7) higher in herds that administered 2 doses of Leptospira vaccine to cows compared to herds that did not vaccinate against this pathogen.

Conclusion and clinical relevance

Based on the serological evidence, few weaning-age beef calves across Canada have been exposed to pathogenic Leptospira bacteria.

Serological evidence of exposure to pathogenic Leptospira has been identified in most mammals and marsupials, including humans (1,2). Historically, the basic units of Leptospira taxonomy are the serovars, which consist of closely related isolates based on serological reactions to the outer membrane lipopolysaccharide (2,3). Leptospira are often classified into 2 major groups: host-adapted serovars, which are adapted specifically to a maintenance host; and non-host-adapted serovars, which can infect multiple incidental hosts (4–6).

The more recent phylogenetic classification of Leptospira, which organizes species based on DNA relatedness, also coexists with the historical serological classification (2,7). Consequently, serovar Hardjo is composed of 2 genetically distinct species that are serologically indistinguishable (6,8). Serovar Hardjo (from either Leptospira borgpetersenii serovar Hardjo genotype Hardjo-bovis or Leptospira interrogans serovar Hardjo genotype Hardjo-prajitno) is the host-adapted serovar for cattle, which can establish asymptomatic chronic renal and genital carriers with long-term shedding of leptospires in urine (5,6).

Transmission between cattle is through direct contact with infected urine or natural mating (3,6). Serovar Hardjo can be difficult to detect in individual cattle, as maintenance hosts generally mount a low or undetectable antibody response to chronic infection (6,9). In cattle, serovar Hardjo infection is more likely to result in early embryonic death, infertility, and sporadic abortions than obvious clinical disease and abortion outbreaks caused by non-host-adapted serovars (3,6).

Leptospira interrogans serovars Bratislava, Canicola, Grippotyphosa, Icterohaemorrhagiae and Pomona are examples of non-host-adapted serovars for cattle. These sporadically infect naïve cattle as incidental hosts, causing acute severe disease with fever, hemolytic anemia, hemoglobinuria, and jaundice, or abortion storms, with short-term urinary shedding of leptospires (3,5,6). Maintenance hosts for the non-Hardjo serovars tested in this study are dogs (Canicola), swine (Pomona and Bratislava), horses (Bratislava), rodents, and wildlife (Grippotyphosa, Icterohaemorrhagiae) (1,2). Cattle can be exposed to non-host-adapted serovars from water, feed, and bedding contaminated with urine from other domesticated or wild animals, with high rainfall facilitating transmission (2–4).

Previous estimates of Leptospira seroprevalence in weaning-age beef calves are based on research that is > 20 y old and focused on western Canada. The 2002 study sampled 1539 unvaccinated beef calves from 61 herds. Only 0.8% of calves from 5 herds had a positive (≥ 100) microscopic agglutination test (MAT) antibody titer to 1 Leptospira serovar, with Hardjo being the most common (4).

The objectives of this study were to estimate the seroprevalence of 6 Leptospira serovars in beef calves from cow-calf herds across Canada at or near the time of fall weaning, and then to estimate the association of between-herd Leptospira vaccination programs and serovar antibody titers in weaning-age calves.

This study was approved by the University Animal Care Committee and Research Ethics Board at the University of Saskatchewan (Saskatoon, Saskatchewan) (Protocol # 2014003). The cow-calf producers in this study were participants in the Canadian Cow-Calf Surveillance Network (C3SN), a national cattle health and productivity surveillance network established in 2018 (10). In the fall of 2021, herd veterinarians collected blood samples from a systematic random sample of up to 20 spring-born calves in each herd. Spring-born calves at or near the time of fall weaning were chosen for sampling because interpretation of serovar titers was unlikely to be complicated by maternal antibodies in this group (11), and calves of this age are often not vaccinated against Leptospira (4), lowering the likelihood of detecting vaccine titers. Following collection, whole blood was sent to Prairie Diagnostic Services (Saskatoon, Saskatchewan) in coolers, via courier, for processing and storage of serum (−80°C) until testing.

Samples were shipped to the Michigan State University Veterinary Diagnostic Laboratory (Lansing, Michigan, USA), an International Leptospirosis Society proficiency tested laboratory. Antibody titers to 6 Leptospira interrogans reference strains representing 6 serovars, Bratislava, Canicola, Grippotyphosa, Hardjo, Icterohaemorrhagiae, and Pomona were measured by MAT following World Organisation for Animal Health-recommended protocols (9). Serum samples were screened at a 1:50 dilution. Samples that produced ≥ 50% leptospire agglutination compared to the control antigen were considered reactive and subjected to 2-fold serial dilutions. Titer end points for each serovar were reported as the reciprocal of the highest dilution of reactive sera demonstrating ≥ 50% leptospire agglutination (e.g., 1:200 = 200).

Leptospira serovar titer data were examined and summarized using a commercial software program (Microsoft Excel; Microsoft Corporation, Redmond, Washington, USA). Microscopic agglutination test titers of ≥ 100 were classified as positive and titers of < 100 were classified as negative (9). Positive herds had ≥ 1 sampled calf with a positive titer to any of the tested serovars.

Vaccine use data for C3SN herds for 2020 had been collected via a mail survey in 2021 (12). Vaccination data for cows in 2020 were used to assess history of vaccination for the dams of calves born in the spring of 2021. Vaccination history for nursing calves in 2020 served as an estimate of vaccine use for nursing calves in 2021.

Generalized estimating equations with negative binomial distribution and log link function were used to estimate the association between Leptospira serovar titers in weaning-age calves and number of doses of Leptospira vaccine (0, 1, ≥ 2) administered to cows, while controlling for herd-level clustering. Titers (0, 50, 100, …, 6400) were converted to a scale reflecting the number of doubling dilutions (0, 1, 2, …, 8). All serovars were included where ≥ 10 calves were positive in total and at least 5 herds had ≥ 1 positive calf. All models were constructed using a commercial software program (STATA version 17.0; StataCorp, College Station, Texas, USA). The relative risk that a serovar titer would increase by 1 doubling dilution and 95% CI were reported.

Serum samples were obtained from 1922 beef calves from 106 herds across the country. Regionally, 1455 calves were sampled from 76 western herds and 467 calves were sampled from 30 eastern herds. Seven participating herds were located in British Columbia, 35 in Alberta, 18 in Saskatchewan, 16 in Manitoba, 13 in Ontario, 13 in Quebec, and 4 in Atlantic Canada. Shipping time to Prairie Diagnostic Services varied from 1 to 4 d, depending on the location of the herd.

At or near the time of fall weaning, 1746 (90.8%) calves sampled from all 106 herds had undetectable (< 50) antibody titers to all 6 Leptospira serovars tested. However, 62 (3.2%) calves sampled from 28 (26.4%) herds had a positive (≥ 100) titer to ≥ 1 serovar. Specifically, 51 (2.7%) sampled calves from 27 herds (25.4%) were positive to 1 serovar, whereas 11 (0.6%) sampled calves from 4 (3.8%) herds were positive to multiple serovars. Two-thirds (19/28) of titer-positive herds had only 1 sampled calf with a positive titer to 1 serovar. Regionally, 30 (2.1%) calves sampled from 20 (26.3%) western herds and 32 (6.8%) calves sampled from 8 (26.6%) eastern herds had a positive titer to ≥ 1 serovar.

The distribution of detectable titer end points varied by serovar (Figure 1). Overall, 176 (9.2%) calves sampled were reactive (titer ≥ 50), 27 (1.4%) had a titer ≥ 400, and 15 (0.8%) had a titer ≥ 1600 to ≥ 1 serovar. The host-adapted serovar Hardjo was the third-most-common serovar detected, with 17 (0.9%) sampled calves having a positive titer (Table 1). Regarding the non-host-adapted serovars, 21 (1.1%), 20 (1.0%), 12 (0.6%), 8 (0.4%), and 3 (0.2%) calves sampled had positive titers to serovars Grippotyphosa, Pomona, Icterohaemorrhagiae, Bratislava, and Canicola, respectively (Table 1).

FIGURE 1.

Distribution of detectable microscopic agglutination test (MAT) antibody titer end points against the host-adapted Leptospira serovar Hardjo and 5 non-host-adapted Leptospira serovars (Bratislava, Canicola, Grippotyphosa, Icterohaemorrhagiae, and Pomona) in serum samples collected at or near the time of fall weaning from 1922 beef calves from 106 Canadian herds.

TABLE 1.

Comparison of the number and proportion of positive (≥ 100) microscopic agglutination test (MAT) antibody titers against the host-adapted Leptospira serovar Hardjo and 5 non-host-adapted Leptospira serovars (Bratislava, Canicola, Grippotyphosa, Icterohaemorrhagiae, and Pomona) in serum samples collected at or near the time of fall weaning from 467 beef calves from 30 eastern Canadian herds versus 1455 beef calves from 76 western Canadian herds.

	Leptospira serovars
	Hardjo	Bratislava	Canicola	Grippotyphosa	Icterohaemorrhagiae	Pomona
Eastern calves n = 467	2 (0.4%)	5 (1.1%)	2 (0.4%)	17 (3.6%)	8 (1.7%)	16 (3.4%)
Western calves n = 1455	15 (1.0%)	3 (0.2%)	1 (0.1%)	4 (0.3%)	4 (0.3%)	4 (0.3%)
Eastern herds n = 30	1 (3.3%)	2 (6.7%)	1 (3.3%)	3 (10.0%)	5 (16.7%)	4 (13.3%)
Western herds n = 76	7 (9.2%)	2 (2.6%)	1 (1.3%)	4 (5.3%)	4 (5.3%)	4 (5.3%)

At the herd level, 8 (7.5%) operations had 1 or more sampled calves with a positive titer to serovar Hardjo (Table 1). Seven of these 8 herds were located in western Canada. Nine (8.5%) herds had 1 or more sampled calves with a positive titer to serovar Icterohaemorrhagiae, 8 (7.5%) to Pomona, 7 (6.6%) to Grippotyphosa, 4 (3.8%) to Bratislava, and 2 (1.9%) to Canicola (Table 1). Three (2.8%) operations had ≥ 3 calves sampled with a positive titer to serovar Hardjo. Three (2.8%) herds had ≥ 3 calves sampled with positive titers to serovar Pomona (2.8%), 1 (0.9%) to Bratislava, 1 (0.9%) to Grippotyphosa and 1 (0.9%) to Icterohaemorrhagiae.

Regionally, at both the calf and herd levels, the seroprevalence of the non-host-adapted serovars was higher in eastern Canada, whereas the seroprevalence of the host-adapted serovar Hardjo was higher in western Canada (Table 1).

Most of the C3SN herds (94/106, 89%) participating in this 2021 seroprevalence study completed a herd-level vaccine use survey for 2020. Cows (34/94, 36%) were much more likely than nursing calves (2/94, 2.1%) to be vaccinated against Leptospira at least once in the past year.

There was no association between weaning-age calf MAT titers for serovars Grippotyphosa, Hardjo, and Pomona and the number of doses of Leptospira vaccine administered to cows. The probability of calf serovar Icterohaemorrhagiae titer increasing by 1 doubling dilution was 3.6× (95% CI: 1.7, 7.7) higher in herds with cows receiving 2 doses of Leptospira vaccine compared to herds that did not vaccinate against this pathogen. There was no significant difference in titers for calves from cows with 1 dose versus no doses of vaccine. As only 2 herds vaccinated nursing calves against Leptospira, it was not possible to estimate the association the between vaccination of nursing calves and serovar titers in weaning-age calves.

This study has provided current estimates of seroprevalence against 6 important Leptospira serovars in weaning-age beef calves from herds across Canada, suggesting limited exposure to leptospirosis. Although overall seroprevalence estimates were low (3.2%), both the calf- and herd-level estimates were higher than those in a 2002 western Canadian study (4). As well, in contrast to the 2002 study, some calves (0.6%) in this study had positive titers to multiple serovars (4).

Convincing serological evidence of herd-level exposure (≥ 3 sampled calves with positive titers) to serovar Hardjo occurred in only 3 herds, all located in western Canada, and none vaccinated against Leptospira. Similar herd-level results for serovar Hardjo were previously reported (4). There was also convincing serological evidence of herd-level exposure to non-host-adapted serovars in 3 herds, all unvaccinated herds located in eastern Canada. Of note, 1 herd had 12 of 17 sampled calves with very high titers (≥ 1600) to serovar Grippotyphosa, suggestive of active Leptospira herd-level infection (2,5). No herds from western Canada had > 1 sampled calf with a positive titer to non-host-adapted serovars, either in the present study or in 2002 (4). Pathogenic Leptospira do not multiply outside the host and require high humidity for survival in the environment (2,3). Given that accumulated precipitation is substantially higher in the beef cattle-rearing areas of eastern Canada (13), the relatively higher seroprevalence of non-host-adapted serovars in eastern Canada was predictable.

In this study, the median age of sampled calves was 7.4 mo (14), with a low likelihood of maternal antibodies against Leptospira still circulating in calf serum (11). Therefore, the absence of an association between Leptospira vaccine administered to cows and weaning-age calf MAT antibody titers for most serovars (Bratislava, Canicola, Grippotyphosa, Hardjo, and Pomona) was not surprising. The association detected between cow vaccination and serovar Icterohaemorrhagiae titers could potentially have been due to a higher frequency of vaccination use in regions with a history of infection and increased likelihood of exposure.

By measuring MAT titers in weaning-age calves rather than in mature cows, the current study may have underestimated the risk of herd exposure. Leptospira exposure in young calves protected by maternal antibodies would be less likely to result in a detectable serological response (11). Furthermore, beef calves widely dispersed on calving and grazing pastures would be at relatively low risk from transmission of serovar Hardjo by either natural breeding or direct contact with a carrier animal’s urine (4,6). Sampled calves had < 8 mo of potential environmental exposure to non-host-adapted Leptospira serovars. Not surprisingly, 2 surveys completed 17 or more years ago demonstrated higher overall seroprevalence to serovars Hardjo, Icterohaemorrhagiae, and Pomona in unvaccinated beef cows (4,15) than in calves in this study, consistent with increased likelihood of exposure over time.

Based on the serological evidence, few weaning-age beef calves across Canada have been exposed to pathogenic Leptospira bacteria. However, the overall low MAT antibody titers observed in weaning-age calves in this study were most likely associated with natural exposure rather than maternal antibodies or vaccination, given both the age of the sampled calves and the very low reported Leptospira vaccine use in nursing calves. CVJ

Funding Statement

Financial support was provided in part by the Beef Cattle Research Council; Saskatchewan Ministry of Agriculture; and Zoetis Clinical Research Funds of the Bovine Ambulatory Clinic (Université de Montréal), for translation services for Quebec producers.