Leptospira

Bacterial strains (Table 1) were cultured by the WHO reference laboratory at the KIT Biomedical Research Centre at The Royal Tropical Institute, Amsterdam, The Netherlands (all isolates and reference strains labelled RK3) and at the Veterinary Sciences Division (VSD), The Queen's University of Belfast, United Kingdom (reference strains labelled RB3) and the WHO reference centre at Port Blair India (labelled isol 15). A total of 120 strains consisting of 79 isolates and 41 reference strains from different sources and geographical regions were analyzed by MLST. The 41 reference strains included in the study belonged to six Leptospira species (L. interrogans; L. kirschneri; L. noguchii; L. borgpetersenii; L. santarosai and L. alexanderi).

Sequence types and the associated sequence data for the seven MLST loci were downloaded from the Leptospira MLST website (http://leptospira.mlst.net) for 199 isolates. MLST was performed using the original scheme for an additional 52 Leptospira isolates that had not been genotyped previously. These included 38 L. interrogans isolates from Laos (n = 11), Malaysia (n = 2), Sri Lanka (n = 14), Japan (n = 1) the Philippines (n = 2), or China (n = 8), and 14 L. kirschneri isolates from Japan (n = 2) or reference collections (n = 12). Alleles were assigned and sequence types defined using the MLST website. The 251 isolates typed by the original scheme are detailed in Table S1.

The Microagglutination Test (MAT), the reference serological test, was used for processing samples, considering a titer of ≥1:200 as a criterion of positivity. This is the cut-off point used in cattle in Argentina since leptospirosis is an endemic disease and because cattle are vaccinated in areas where this agent is present (17 ). Antibodies against pathogenic Leptospira were detected by the MAT in the Leptospirosis Laboratory, Department of Rural Zoonosis (Ministry of Health of Buenos Aires Province), according to WOAH (18 ) protocols.
A panel of live antigens of ten Leptospira spp. reference strains were used: L. interrogans serogroup Canicola serovar Canicola strain H. Utrecht IV, serogroup Hebdomadis serovar Hebdomadis strain Hebdomadis, serogroup Icterohaemorrhagiae serovar Copenhageni strain M20, serogroup Pomona serovar Pomona strain Pomona, serogroup Pyrogenes serovar Pyrogenes strain Salinem, serogroup Sejroe serovar Wolfii strain 3,705 and serogroup Sejroe serovar Hardjo strain Hardjoprajitno, L. borgpeterseni serogroup Ballum serovar Castellonis strain Castellon 3 and serogroup Tarassovi serovar Tarassovi strain Perepelitsin and L. kirschneri serogroup Grippotyphosa serovar Grippotyphosa strain Castellon 3. This panel was developed at 28–30°C in the Ellinghausen-McCullough-Johnson-Harris (EMJH) medium with no more than 15 days of growth. Serial serum dilutions were performed with phosphate-buffered saline (PBS, pH 7.2) starting from 1:100 dilution. The plates were incubated at 37°C for 90 min. After incubation, the serum-antigen mixtures were checked for agglutination under a dark field microscope. Tests were interpreted as positive when agglutination at ≥ 1:200 of at least 50% of the leptospires for any serogroup was observed. The highest serum dilution with >50% agglutination or ≤50% free leptospires, compared to the negative control, was considered the endpoint titer of quantitative MAT.

The data about each animal, the characteristics of each farm and laboratory results were entered into an Excel database (Microsoft, Redmond, WA, USA). Prevalence of anti-Leptospira spp. antibodies with the 95% Confidence Interval (CI) was estimated. Also, farms positivity was determined. The association between the outcome seropositivity to Leptospira spp., to Leptospira serogroup Sejroe (adapted serogroup) and to Leptospira serogroup Pomona (incidental serogroup) and the variables under analysis was assessed by a bivariate analysis using a Chi-squared test. Fisher's exact test was used if one or more cells expected value was <5. Odds ratios (OR) and 95% CI were also estimated and calculated for each variable. For quantitative variables, parametric or non-parametric tests were used. The null hypothesis was that there were no differences between groups. All the statistical tests were carried out at α=0.05. Quantitative and qualitative variables at p-value < 0.2 in the bivariate analysis were analyzed by a Generalized Linear Mixed Model (GLMM) with random effect of farm-level risk. All statistical analyses were performed with software R v. 4.0.2 (10 ).
Potential spatial clusters were investigated in the study area with space scan statistics using SaTScan software, v10.0.2. Poisson model for high rates was performed for detecting spatial patterns of the number of MAT positive events in a geographical location, taking each farm as a unit for analysis according to a known population at risk (19 (link)).
Another qualitative variable called “being inside the spatial clusters” was generated, and an animal was considered to be exposed to the variable when it belonged to a farm inside a significant cluster. The animal was not exposed to the variable when it belonged to a farm outside the significant clusters. GLMM was performed with the significant variables detected in the first GLMM with a new variable, “being inside the spatial clusters.”

A cross-sectional serological survey was carried on from September 2017 to December 2020. Using a probabilistic two-stage sampling, 25 farms and fifteen animals per farm were selected. SENASA (Servicio Nacional de Sanidad y Calidad Agroalimentaria) and Fundación Aftosa provided the region's database. In the first stage, the number of farms to be selected was calculated using the ProMesa 1.3 programme (12 ) using the following formula:
Where p is the expected seroprevalence of cows reactive to Leptospira, z is the confidence level, ROH is the homogeneity rate, b is the number of animals selected per farm and e is the acceptable (allowable) absolute error. Our assumptions were as follows: a prevalence of 20.33% (13 ), a relative error of 33%, a low homogeneity rate (0.06–0.12) (14 (link)) and 15 animals sampled per farm. The minimum sample size estimated was 25 farms (375 animals). More farms were included from Ayacucho (14) because this department has the largest number of cattle farms. The second stage was conducted on each farm through systematic randomization of the animals. The animals included in the study were bulls and breeding females ≥15 months of age, that were not vaccinated against Leptospira spp. or had been vaccinated more than 6 months before sampling. In addition, a questionnaire was administered to collect epidemiological information about the potential risk factors associated with seropositivity to Leptospira spp. The survey was based on general production and environmental characteristics, feeding practices, health and reproduction status. All farms were georeferenced using Global Positioning Systems (GPS) to perform spatial analysis and subsequent search for meteorological and satellite information such as the presence of water bodies, potential evapotranspiration, temperature and solar radiation. Water bodies were detected using information from the Landsat 8 satellite (spatial resolution of 30 meters) and calculated with MNDWI (Modified Normalized Difference Water Index) (15 (link)) with the QGIS software. The potential evapotranspiration was obtained through the EOS-Terra satellite and estimated using the ENVI software. The temperature was calculated monthly through data from the National Meteorological Service and solar radiation, using information from the sensor CERES (Clouds and the Earth's Radiant Energy System) (16 (link)). On the farms, blood samples were collected from the animals by venipuncture. The blood was allowed to clot at room temperature and centrifuged at 1,500 rpm for 15 min. The sera were separated and stored at −20°C.