Diagnostic accuracy of an immunomagnetic separation-PCR assay to detect pathogenic Leptospira spp. in urine from dairy cattle, using a Bayesian latent class model.

Leptospirosis is a zoonotic disease that has spread worldwide and causes significant economic losses in the dairy industry. The causal agents of this infectious disease are members of the genus Leptospira, known as pathogenic Leptospira spp. Specific clinical signs of the infection are difficult to detect. Therefore, the disease is normally under-diagnosed, mostly due to the lack of a cost-effective technique for diagnosing animals with a low bacterial load in their urine. The aim of this study was to assess the diagnostic accuracy of a qPCR coupled with a previous Immunomagnetic separation (IMS) step (IMS-qPCR) against a qPCR without using IMS, using a Bayesian latent class model (2 tests, 3 populations) to determine the leptospirosis infectious status in naturally infected dairy cattle. The results revealed that IMS qPCR had a sensitivity (Se) of 95.7% (95% Probability Interval (PI) = 85.0; 99.4%) and a specificity (Sp) of 98% (95% PI = 96.1; 99.4%), indicating that it is more sensitive than conventional qPCR (Se = 69.7% (95% PI = 59.2; 79.0%); median difference = 25.2% (Monte Carlo Error = 10.2%); and the Sp = 98.8% (95% PI = 97.6; 99.5%), median difference = 0.8% (Monte Carlo Error = 2.1%). Therefore, results shows that IMS-qPCR is a more useful diagnostic tool in terms of accuracy for detecting infectious animals with pathogenic Leptospira in their urine.

Development and evaluation of polyclonal antibody-based antigen detection ELISA and dot blot assays as a less costly diagnostic alternative for pathogenic Leptospira infection.

Leptospirosis is an infectious, zoonotic disease of worldwide distribution, the cause of which is infection by pathogenic Leptospira. In Chile, dairy cattle are recognized a significant source in the maintenance and transmission of this infection, which causes economic losses and represents an infection threat to workers in the dairy industry. The infection is underestimated in cattle, due to the lack of clinical, pathognomonic signs, as well as the low efficiency of current diagnostic techniques. In this study, we developed antigen ELISA and dot blot assays, based on polyclonal antibodies, to detect pathogenic Leptospira in the urine samples of dairy cattle. The proposed tests showed an acceptable diagnostic accuracy, based on an analytical sensitivity of 1·104 Leptospira per mL for ELISA, and 3.2·103 for dot blot. These results corresponded with those obtained by qPCR, and the use of urine samples allowed us to propose new diagnostic alternatives for pathogenic Leptospira infection at a low cost, which can provide information on active infection status, which is a key element in control programs both at individual and herd level.

Association between the severity of histopathological lesions and Mycobacterium avium subspecies paratuberculosis (MAP) molecular diversity in cattle in southern Chile.

The objective was to evaluate the association between the severity of histopathological lesions caused by Mycobacterium avium subspecies paratuberculosis (MAP) infection and the molecular diversity of this pathogen. Blood, ileum, and mesenteric lymph node samples were collected at slaughter, from 1,352 adult cattle [source population 1 (SP1)]. In addition, 42 dairy herds (n = 4,963 cows) were followed for 2 years, and samples from compatible paratuberculosis clinical cases [source population 2 (SP2)] were collected. MAP infection was confirmed using an ELISA test, liquid media culture, and PCR. Isolates were genotyped using five MIRU-VNTR markers. Tissues from confirmed samples were subjected to a histopathological examination. A histopathological severity score (HSS) system was developed and used to grade (0 to 5) the magnitude of lesions caused by MAP. In general, the HSS system assesses the number of foci and degree of macrophage infiltration, together with the presence of multinucleated giant cells (MGCs) and acid-fast bacilli (AFB), in addition to the fusion of the intestinal villi and hyperplasia of the crypts. Despite the large sampling effort, only 79 MAP isolates were successfully genotyped, where 19 different haplotypes were described. A mixed-effect Poisson regression model was used to assess the relationship between haplotypes and HSS values. The model was controlled by animal age, and the farm was used as a random effect. Haplotypes were grouped based on their relative frequency: the most frequent haplotype (group i, 49.4%), the second most frequent haplotype (group ii, 12.7%), and all other haplotypes (group iii, 37.9%). Model outputs indicated that group i had significantly higher HSS values than group iii. In addition, group i was also associated with higher optical density (OD) values of the ELISA test. These results support the existence of differences in pathogenicity between MAP haplotypes. However, results were based on a relatively small sample size; thus, these should be taken with caution. Despite this, study findings suggest that haplotypes would be associated with differences in disease progression, where the dominant haplotype tends to generate more severe lesions, which could be linked to a greater shed of MAP cells than non-dominant haplotypes, increasing their chances of transmission.

Analytical evaluation of an immunomagnetic separation PCR assay to detect pathogenic Leptospira in cattle urine samples obtained under field conditions.

Clinical manifestations of leptospirosis are diverse and very similar to other febrile diseases, hence early and accurate detection of subclinical infections is a key element in disease control. We evaluated immunomagnetic separation (IMS) capture technology coupled with a standard quantitative PCR (qPCR) system for the detection of pathogenic Leptospira in urine samples from 803 cows from dairy herds with a history of clinical cases of leptospirosis. The urine samples were first processed in a purification step, then subdivided into 2 subsamples, one that continued to DNA extraction and direct qPCR, and one that was pretreated by IMS before continuing to DNA extraction and qPCR. Overall, 133 of 803 (16.6%) samples were IMS-qPCR positive, whereas only 92 of 803 (11.5%) were positive when using direct qPCR. Statistically significant differences were observed between the mean estimated Leptospira load between the IMS-qPCR and the direct qPCR positive urine samples. The IMS-qPCR technology revealed a larger number of positive results and higher bacterial loads than direct qPCR. This difference is most likely the result of the high antigen-binding capacity and capture efficiency of the IMS system. The use of polyclonal antibodies produced by the inoculation of 3 synthetic peptides, which make up the extracellular regions of the LipL32 protein, provided a high detection capacity to the IMS-qPCR technique, resulting in performance superior to direct qPCR.

New insights on the infection of pathogenic Leptospira species in American mink (Neovison vison) in southern Chile.

Leptospirosis is a zoonosis of global distribution, caused by the infection of pathogenic Leptospira, a group of bacteria capable of infecting both domestic and wild animals. Mink (Neovison vison) in southern Chile is recognized as a wild and synanthropic rodent predator (among various other prey), and Leptospira infection in them can be acquired through contact with the pathogen in the environment or by eating infected prey. Thus, the aim of this study was to provide more specifics regarding the source of the infection for the American mink under the conditions of Southern Chile. Minks were captured in the Los Ríos region, southern Chile, in an area with well-developed dairy farming. Two areas were selected for mink trapping, one with a high degree of dairy farming and a second with a low degree of dairy farming. Within them, 16 study sites were visited, and 45 American mink were trapped and euthanized to obtain kidney tissue and blood serum samples for bacteria isolation and determination of antibodies titers, respectively. Molecular characterization of the isolated strains was performed. Three minks from sites of high-dairy farming industry and only one from sites with low-degree dairy farming were detected as infected through molecular confirmation. This study shows evidence that confirms previous findings made in southern Chile, regarding mink as host of Leptospira interrogans serovar Hardjo-prajitno associated to cattle-farming areas. However, typing information ( Leptospira interrogans Copenhageni and Icterohaemorrhagiae ) suggests that the consumption of rodents may also be a potential source of infection.