A serological survey of canine leptospirosis in the city of Belgrade, Serbia

Canine leptospirosis is a zoonosis caused by bacteria belonging to the genus Leptospira. Dogs are one of the animal species involved in the cycle of preservation and transmission of leptospirosis in urban areas. Serological testing for the presence of specific antibodies against Leptospira spp. in dogs was continuously performed between 2010 and 2020 in the city of Belgrade. At the request of the owners themselves, other veterinary laboratories or laboratory clinics, 179 blood sera from 179 dogs were examined in the Laboratory for Immunology, Scientific Institute of Veterinary Medicine of Serbia. Blood sera samples from dogs were examined using the standard microscopic agglutination test (MAT) for the presence of specific antibodies against seven different serovars of Leptospira: Pomona, Icterohaemorrhagiae, Grippotyphosa, Sejroe, Canicola, Bataviae, and Australis. The number of seropositive dogs was 17/179 (9.5%). Among all examined sera, the highest titre of seropositive samples was to serovar Icterohaemorrhagiae (10/17, 58.8%), followed by Pomona (4/17, 23.5%), and serovar Canicola (3/17, 17.6%). Specific antibodies for serovars Grippotyphosa, Sejroe, Bataviae and Australis were not detected in any of the dog sera. Cross-reaction (the presence of two or three titres with different values where one of them was higher than others) between different serovars was diagnosed in a low number of sera (n=4), with the following serovars: Icterohaemorrhagiae and Pomona (n=3) and Pomona and Canicola (n=1). The confirmed specific antibody titres for Leptospira spp. were between 1:100 to 1:3000 (5 sera had titres of 1:100, 7 had titres of 1:300, 4 had titres of 1:1000, and 1 serum had a titre 1:3000). Monitoring canine leptospirosis is a useful tool in preventing leptospirosis in Belgrade.

Evaluation of Real-Time Polymerase Chain Reaction in Culture-Negative Cerebrospinal Fluid Samples of Bacterial meningitis Patients

Background: Meningitis is a rigorous childhood disease with high morbidity and mortality. It is the main cause of under five mortality in India. Mainly three bacteria Streptococcus pneumoniae, Neisseria meningitidis, and Haemophilus influenzae are responsible. In low economic set up country like India, documented bacterial meningitis mainly depend on gram staining, cerebrospinal fluid (CSF) culture results or latex agglutination test resulting in less number of positive due to the prior antimicrobial intake which affects culture and latex agglutination test results. This study was taken up rapid and accurate molecular method like RT PCR to diagnose bacterial meningitis in culture-negative CSF samples. Materials and Methods: Fifty culture-negative CSF samples from suspected cases of bacterial meningitis were examined by real-time polymerase chain reaction (real-time PCR) for the presence of lytA, bexA, and ctrA genes specific for Streptococcus pneumoniae, Haemophilus influenzae, and Neisseria meningitidis respectively. Results: Positive real-time PCR results for Streptococcus pneumoniae were detected in 36 (72%) of culture-negative CSF samples while 10% positive results for Haemophilus influenzae type b. Nine (18%) samples were negative by real-time PCR for all tested organisms. Conclusion: The use of molecular techniques as real-time PCR can provide a valuable addition to the proportion of diagnosed cases of bacterial meningitis especially in settings with high rates of culture-negative results.

Epidemiological situation of human leptospirosis in Brazil and challenges in its diagnosis with a focus on molecular approaches

Leptospira interrogans is one of the causative agents of human leptospirosis, a zoonotic disease with worldwide distribution. Nowadays, this zoonosis is considered one of the biggest in terms of morbidity and mortality (even considering Dengue, the major arbovirosis affecting humans), having in Brazil 3,800 human cases per year. Currently, difficulties imposed by the absence of a rapid, sensitive diagnostic test that can be used as a routine test for the detection of leptospirosis lead to misdiagnosis and underreported cases. The gold standard diagnostic test for leptospirosis is the microscopic agglutination test (MAT), which presents difficulties in execution and interpretation. Therefore, this review proposes a general view of the epidemiologic situation of the disease in Brazil, in addition to the current contributions in the literature for the development of new diagnostic methods. Amongst them, the gene sequences polymorphism analysis, which presents potential for phylogenetic and populational analysis and genotyping of Leptospira spp.

Serologic titers to Leptospira in vaccinated pigs and interpretation for surveillance

Diagnosis and surveillance of pathogenic Leptospira is difficult as organisms may be intermittently shed and in small numbers. Therefore, serologic testing by the microscopic agglutination test (MAT) is the primary screening method for leptospirosis. While a MAT titer ≥1:100 is considered to be a positive result, interpretation is complicated by the use of commercial vaccines in pigs. Most guidelines for interpretation of MAT titers in pigs were published in the 1970’s and 1980’s, prior to the development of the current multivalent vaccines. We evaluated MAT titers in routinely vaccinated healthy research pigs compared to their unvaccinated cohorts. Our study confirmed previous reports that the Pomona serovar elicits minimal antibody response even after a second booster 6 months after initial vaccination. However, MAT titers of ≥1:3,200 were detected as early as 4 weeks post initial vaccination for serovars Bratislava and Icterohaemorrhagiae and remained as high as ≥1:1,600 prior to booster at 24 weeks post vaccination. Our study determined that high levels of MAT titers can occur from vaccination alone and high titers are not necessarily indicative of infection. Therefore, the interpretation of MAT titers as indicators of Leptospira infection should be readdressed.

A machine learning model of microscopic agglutination test for diagnosis of leptospirosis

Leptospirosis is a zoonosis caused by the pathogenic bacterium Leptospira. The Microscopic Agglutination Test (MAT) is widely used as the gold standard for diagnosis of leptospirosis. In this method, diluted patient serum is mixed with serotype-determined Leptospires, and the presence or absence of aggregation is determined under a dark-field microscope to calculate the antibody titer. Problems of the current MAT method are 1) a requirement of examining many specimens per sample, and 2) a need of distinguishing contaminants from true aggregates to accurately identify positivity. Therefore, increasing efficiency and accuracy are the key to refine MAT. It is possible to achieve efficiency and standardize accuracy at the same time by automating the decision-making process. In this study, we built an automatic identification algorithm of MAT using a machine learning method to determine agglutination within microscopic images. The machine learned the features from 316 positive and 230 negative MAT images created with sera of Leptospira-infected (positive) and non-infected (negative) hamsters, respectively. In addition to the acquired original images, wavelet-transformed images were also considered as features. We utilized a support vector machine (SVM) as a proposed decision method. We validated the trained SVMs with 210 positive and 154 negative images. When the features were obtained from original or wavelet-transformed images, all negative images were misjudged as positive, and the classification performance was very low with sensitivity of 1 and specificity of 0. In contrast, when the histograms of wavelet coefficients were used as features, the performance was greatly improved with sensitivity of 0.99 and specificity of 0.99. We confirmed that the current algorithm judges the positive or negative of agglutinations in MAT images and gives the further possibility of automatizing MAT procedure.

Serological prevalence of Leptospira serovars among pigs in Ukraine during the period of 2001–2019

Leptospirosis is a widespread infection among pigs throughout the world. In most cases in Ukraine, only the microscopic agglutination test (MAT) is used for the diagnosis of leptospirosis in animals. In general, during the period of 2001–2019, 2 381 163 samples of blood sera from swine were tested in our country and 85 338 positive reactions were received, which is 3.58% [binomial confidence intervals (BCI), 3.56–3.61%]. It was established that the serovars copenhageni – 33.91% (BCI, 33.59–34.23%), bratislava – 14.14% (BCI, 13.90–14.37%), pomona – 8.58% (BCI, 8.39–8.77%), and tarassovi – 7.12% (BCI, 6.95–7.30%) play a leading role in the aetiological structure of swine leptospirosis. A large number of positive reactions to several serovars was observed – 29.78% (BCI, 29.47–30.09%) of the total number of positive cases. In addition, the article presents data according to a retrospective analysis of the eight serovars circulating among pigs in Ukraine. Thus, during the nineteen year period, there was a decrease in the number of positive reactions to bratislava, pomona, and tarassovi and an increase in the number of positive reactions to copenhageni, polonica, and kabura. Mapping Ukraine’s territory for leptospirosis among pigs was carried out. This allows one to identify zones with a risk of leptospirosis infections among swine. The maps show that the highest incidence rates were identified in the eastern and central parts of Ukraine.

DETECTION OF TOXOPLASMOSIS IN CATS AND SHEEP

Thirty-three fecal samples from cats were examined for the presence of Toxoplasma oocysts, and another 33 serum samples from these cats were subjected for Latex agglutination test & indirect immunofluorecent antibody test. Also 80 serum samples from ewes were subjected to the same serological tests. The study indicated that the prevalence of Toxoplasma oocysts in cats was 27.3%. Higher rates of antibody titer (68%) were observed in cats tested with latex test. Infection in young cats was higher than in adults. Sixty percentage of ewes were sero-positive with Latex test, but only 35% were sero-positive with IFAT, higher prevalence of antibody titers was observed in sheep from the three locations of Iraq. Ewes that had recurrent abortion showed higher prevalence in both tests than non aborted ewes.

Detection of Anti-LipL32 Antibodies in Serum Samples from Horses with Chronic Intraocular Infection with Leptospira spp.

Equine recurrent uveitis (ERU) is typically caused by chronic intraocular leptospiral infection in warm-blooded horses in central Europe. The most effective therapy for leptospiral-induced ERU is the surgical removal of diseased vitreous (vitrectomy). Since vitrectomy is a highly specialized and invasive surgery, the indication must be determined very carefully. In order to obtain evidence of intraocular leptospiral infection by laboratory diagnostics in questionable leptospiral ERU-cases, sampling of aqueous humor is required, because serum tests using microscopic agglutination test (MAT) are too unspecific. The SNAP Lepto is a cross-species rapid test for the detection of anti-Lipl32 antibodies that has a high sensitivity (0.97) and specificity (1.00) for the detection of anti-leptospiral antibodies using aqueous humor or vitreous samples, which is comparable to MAT. To evaluate sensitivity and specificity of SNAP Lepto using serum, serum samples from 90 horses with confirmed leptospiral ERU and from 103 ocularly healthy horses were tested by both MAT and SNAP Lepto. Sensitivity was similar for both tests (0.82 vs. 0.79), but specificity was lower for MAT (0.52 vs. 0.95). Sensitivity and specificity are therefore lower in serum samples compared to intraocular samples, however, the SNAP Lepto is far superior to MAT and suitable as a screening method using equine serum.

Physical, Biochemical, Cytological, Bacteriological Screening of Carpal hygroma Fluid vis-a-vis Surgical Management of Carpal hygroma in Cattle and Buffaloes- A report of 15 Cases

Fifteen animals affected with unilateral 9 and bilateral 6 carpal hygroma were presented with the history of swelling on the anterior aspect of the carpus since 3-18 month. Hygroma fluid samples from all the cases were aseptically collected for physical, biochemical, cytological and bacteriological investigations. The colour of hygroma fluid was pale yellow with slight deposits. Glucose, chloride and total protein levels were 43±4.08 mmol/dL, 107±6.50 mmol/dL and 3.20 g/dL, respectively. Cytological examination revealed cell count of 150 cells/µL, 20% neutrophils and 80% lymphocytes. Moreover, mild (+) cellular degeneration changes were also seen. The hygroma fluid samples from all animals were screened for brucellosis. Two cows were found positive for Brucella in tube agglutination test with antibody titres of 160 and 320 IU, respectively. Whereas, the hygroma fluid samples from other animals showed no growth on culture. Surgical excision of carpal hygroma in all cases was done under xylazine sedation (@ 0.02mg/kg body wt) and intravenous regional anaesthesia (IVRA). In brucella infected cows (n=2), hygroma sacs were excised en mass. Skin sutures were applied and the limb was put in fiber-glass cast for 10 days. In bilateral cases, the hygroma of one limb was treated at first instant followed by counter limb after 15 days. In all cases, the wound healing occurred by first intention without any complication. It was concluded that brucella organism may be present in hygroma fluid and due precaution are required while collecting fluid samples. The presence of hygroma may be considered as evidence of brucellosis in the herd. The owners should be advised not to breed such animals in future.