[Basis for the possible use of the indirect immunofluorescence reaction for the serological diagnosis of tick-borne borreliosis].
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Restriction endonuclease analysis of DNA of Leptospira interrogans, serovar hardjo, showed two distinct types within this serovar. These two types, hardjoprajitno and hardjobovis, cannot be differentiated by monoclonal antibodies. Application of 32P- or biotin-labelled total DNA probes in dot-blot or in situ hybridization assays showed a high sensitivity of the assays but also considerable cross-hybridization. Therefore, a genomic library of hardjobovis was constructed and a number of hardjobovis-specific recombinant clones were isolated. Finally, four clones were selected on the basis of a strong hybridization signal and a high specificity for hardjobovis as compared to hardjoprajitno. In a dot-blot assay as well as in in situ hybridization experiments all four clones gave strong signals, and no cross-hybridization with hardjoprajitno was observed in either type of assay. Our results indicate that specific recombinant DNA probes might provide tools for routine diagnosis and classification in cases of hardjo infections.
The recombinant leptospiral protein LipL32 was evaluated for use in the diagnosis of bovine leptospirosis by enzyme-linked immunosorbent assay (rLipL32 IgG ELISA). The microscopic agglutination test (MAT) of 150 serum samples from cattle suspected of leptospirosis showed that 125 (83.3%) samples had positive reciprocal agglutination titres, which ranged from 100 to 1600. The highest titres were observed for the serovars Hardjoprajitno and Bratislava. In the rLipL32 IgG ELISA, 83.3% of the samples were positive. The sensitivity of IgG ELISA for 125 bovine sera, which had MAT titres of greater than or equal to 100, was 100%. ELISA showed a specificity of 100% with 58 bovine sera, which were negative at a 1:50 dilution in the MAT for Leptospira interrogans serovars. When analytical specificity of the IgG ELISA was evaluted using 60 bovine serum samples from animals showing serum antibodies to other pathogens that cause abortion in cattle, such as Babesia sp., Anaplasma sp. and Brucella sp. and no cross-reaction was observed. The recombinant LipL32 IgG ELISA can be an alternative to the MAT for diagnosis of leptospiral infection in cattle.
Between the dates of May 4th-August 6th 2002, 46 cases were detected with abdominal pain nausea, vomiting, arthralgia/myalgia, headache, fever, diarrhea and rash, in the middle Blacksea and north inner Anatolia regions. Their laboratory findings yielded elevated levels of liver enzymes (AST, ALT, LDH), leucopenia and thrombocytopenia. As the infection was treated easily with tetracyclines, clinical diagnosis was considered to be rickettsiosis or ehrlichiosis. Serum and blood samples obtained from some of the patients were tested against Rickettsia, Ehrlichia, Leptospira and Coxiella, in the national and international laboratories. Samples from 19 patients were sent to National Reference Centre and WHO Collaborating Centre for Rickettsial Reference and Research Laboratory, France, and 7 of them were reported as acute Q fever while 8 of them were reported as passed Q fever (QF) cases. In May 2003, new cases with similar symptoms have been reported from the same regions, with different epidemiologic and serologic findings (tick exposure history was higher, response to tetracycline was lower, C. burnetii antibodies were negative), indicating a viral etiology. The samples of these patients have been sent to National Reference Centre and WHO Collaborating Centre for Arboviruses and Viral Heamorrhagic Fevers, France, and the initial reports were marked as Crimean Congo hemorrhagic fever virus (CCHFV). Then the serum samples of previous 26 patients which were stored in National Serum Bank have been retrospectively investigated for viral aetiology in the same center, and 17 of them have been found positive for CCHFV IgM antibodies. Four of these patients were diagnosed as acute QF in 2002, one was passed QF, 2 were negative for QF and 10 were patients not investigated for QF. As a result, the detection of the both infections together in the same area shows the essential need for further epidemiological investigations.
A novel antigenic protein of pathogenic Leptospira, Loa22, was identified by using the PhoA fusion method followed by immunoblotting with convalescent mouse sera. Loa22 was shown to be a lipoprotein having a C-terminal OmpA consensus domain. Loa22 was detected among pathogenic leptospires but not among non-pathogenic leptospires, suggesting the possible involvement of this protein in virulence. The results of three different experiments suggested that Loa22 is located in the outer membrane and a small portion is exposed on the cell surface. Thus, Loa22 may be a candidate for a novel vaccine against infection with pathogenic leptospires.
A dipstick assay for the detection of Leptospira-specific immunoglobulin M (IgM) antibodies in human sera was evaluated in 27 laboratories in 23 countries. 873 serum samples from 711 patients including 329 laboratory-confirmed leptospirosis case patients, 239 noncase patients and 69 patients with viral infections causing heamorrhagic fever were tested. Relative to the results of the reference leptospirosis test, the sensitivity of the dipstick assay was 84.5% for serum samples collected during the first 10 days of the disease and 92.1% for serum samples collected 10-30 days after the onset of disease. The specificity was 87.5% and 94.4%, respectively. Similar to viral haemorrhagic fevers, leptospirosis may cause bleeding. A small number of serum samples from patients with haemorrhagic viral infections gave a weak (1 +) stain. All other samples were negative. In conclusion, the dipstick assay is sensitive and specific and reacts well with serum samples from patients infected with a range of leptospiral strains. It is also easy to use and does not require special equipment or refrigeration. Therefore the assay is ideal for use in developing countries and rural settings.
Investigations were made to determine the causal agent of an acute outbreak of abortions recorded in a swine herd in Mozambique. Isolation of porcine parvovirus and demonstration of its specific antibodies accomplished by using enzyme-linked immunosorbent assay, haemagglutination inhibition and immunofluorescent tests, indicated that porcine parvovirus was the causal agent of the abortions. Other pathogenic agents causing reproductive failure, e.g. pseudorabies virus, Leptospira or Brucella species, were ruled out because investigations of these agents proved to be negative.
Percentage of serological positivity examined in 4205 blood sera by serological method microscopic agglutination test (MAT) on the hinterland territory of our laboratory (East Bohemia; 1999-2003) was 0.38-4.7 %. By the PCR method for detection of DNA of pathogenic leptospires (L. interrogans, L. borgpetersenii and L. kirschneri) from 57 samples of different biological materials from patients with fever of unknown etiology positive results were obtained in 4 specimens (7 %; 3 samples of urine and 1 sample of blood). This method was shown to distinguish between pathogenic and nonpathogenic strains and can detect 2.5-10 cells per mL of biological material. As an important presumption of successful detection of pathogenic leptospires a correct collecting of blood, urine samples or liquor is required before starting antibody therapy. The PCR method possesses a clear advantage over other methods, such as MAT, which relies on the detection of antibodies the presence of which cannot be detected until days after infection.
Infertility is the diminished or absent capacity to produce viable offspring. Infections that reduce ovulation rates, fertilization rates, embryonic survival rates, fetal survival rates or perinatal survival rates result in observed infertility in beef cows. Reproductive pathogens include Leptospira, Campylobacter, Hemophilus, Brucella, bovine herpesvirus-1, bovine viral diarrhea virus, Tritrichomonas foetus, and Neospora caninum. Infectious infertility can be prevented or controlled with appropriate surveillance, biosecurity, and/or vaccination. The objective of this review is to briefly summarize current scientific information to assist with adoption of surveillance methods, implementation of biosecurity and selection of appropriate commercially available vaccines.
In 1970-1979, the incidence of human leptospirosis in Israel was 0.7 per 100,000 population. The majority of the cases (62%) occurred in northeastern Israel (Upper Galilee). Prior to 1973 the main infecting serotypes were grippotyphosa (41%) and Hebdomadis szwajizak (31%). Following the first outbreak of Hebdomadis hardjo infection in 1973, a change occurred in the epidemiologic pattern of human leptospirosis, with hardjo becoming the most common serotype (59%). Hardjo infection outbreaks were sporadic and localized to dairy farms. The peak of incidence was during the summer months, June-September. All the patients with hardjo were dairy workers. The illness was relatively mild and mostly unicteric. Cattle seemed to be the principal source of hardjo infection for man.
Sheep (3918) from 137 farms in the regions of North-, West- and Mid-Netherlands and Gelderland were serologically investigated for the presence of antibodies against Leptospira hardjo. Antibodies were detected in 3.3% of the sheep. There were large regional differences with respect to both the percentage of positive sheep and the percentage of positive flocks. All sera from sheep in Gelderland were negative. In West- en Mid-Netherlands there were 0.9 and 6.5% positive sera, respectively, and 19.4 and 32.7% positive flocks. The percentage of positive sheep per positive flocks varied from 1 tot 51.6. Serological positive sheep were from farms with and without cattle. On farms with cattle, there was no clear relationship between serologically sheep and the presence of hardjo antibodies in cattle. It thus seems plausible that sheep can be infected with hardjo independently of cattle.
Leptospira interrogans serovars pomona, hardjo and tarassovi were each used to inoculate 6 cattle. Three-hundred and ninety-nine sera collected from the inoculated animals and from a control group over a 3-month period were tested using the microscopic agglutination test (MAT) and the enzyme-linked immunosorbent assay (ELISA). Leptospiruria was monitored by microscopic examination and culture. The ELISA detected specific IgM antibody against the serovars in all infected cattle 1 week after inoculation. This IgM antibody persisted in most of the animals for 3-5 weeks. Specific IgG antibody appeared at the same time or just after IgM, but persisted for much longer. Levels of antibody detected by the ELISA and the MAT did not correlate with each other, nor with the periods of leptospiruria found in the infected cattle.
Eighteen patients involved in a localized outbreak of leptospirosis were subjected to a serological follow-up study over a 5-year period. Four distinct sets of sera from all patients and a fifth sample obtained from 10 of them were examined by the microscopic agglutination test (MAT) for demonstration of leptospiral antibodies. The test was carried out by using live leptospires from reference strains of 17 Leptospira interrogans serovars known to occur in Italy. In all cases, the highest titers of agglutinins were recorded against one or more of the three Australis group serovars tested (australis, bratislava, and lora). The highest antibody levels were reached soon after the acute phase of infection in some patients but only after some months in others. Titers then tended to recede with varying rapidity, but titers against the Australis group serovars were still detectable in some patients after 5 years. Coagglutinins against serovars of other serogroups were detected, generally at low levels, in the early sets of sera of most patients, but tended to disappear in the late-set sera. Specific immunoglobulin M (IgM) and IgG against the three Australis group serovars were determined in most serum samples from 16 patients by solid-phase enzyme immunoassay (EIA). In general, EIA titers were considerably lower than MAT titers, but there was a certain patient-to-patient variability in both the IgM/IgG ratio and the evolution and persistence of the two immunoglobulin classes. Since all the evidence indicated that the initial outbreak from a single source, the observed patient-to-patient variability in the progress of both MAT and EIA titers appeared to be attributable to factors inherent in the individual patients. Cross agglutination absorption tests, aimed at retrospectively determining to which of the Australis group serovars the outbreak-specific infecting strain belonged, were performed with six serum samples from different patients. Most absorbed sera seemed to originate from an australis or lora infection, but it was not possible to discriminate conclusively between the two serovars.
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As with most liver diseases, the symptoms of hepatitis in dogs are nearly always aspecific: the dogs eat less, are apathetic, sometimes have polyuria/polydipsia, and sometimes have diarrhoea. Hepatoencephalopathy and ascites only occur with these symptoms in very advanced stages of chronic hepatitis. Only a part of the dogs have jaundice. Because of these aspecific symptoms, the diagnosis hepatitis is often not taken into consideration, even though the presence of a liver disease can be easily detected by measuring plasma concentrations of alkaline phosphatase and bile acids, one or both of which are elevated. The diagnosis is confirmed by histological examination of a liver biopsy sample. The most common forms of hepatitis are non-specific reactive hepatitis, acute hepatitis, and chronic hepatitis. Non-specific reactive hepatitis is a reaction against endotoxin as a result of sepsis or an increased gastrointestinal absorption. Treatment is directed to the primary process. Leptospirosis also causes non-specific reactive hepatitis, but then renal insufficiency is the most prominent feature. The diagnosis is made not on the basis of a liver biopsy but on the basis of increased IgM titres against Leptospira. Immediate treatment with antibiotics and infusions at the first signs (jaundice and uraemia) can save the animal's life. Acute hepatitis can develop as a result of infection, toxins, or liver hypoxia. There is no specific treatment, but adequate recovery often occurs with supportive treatment. Corticosteroids are contraindicated. Chronic hepatitis, which can lead to cirrhosis, is the most common form of hepatitis. It is an autoimmune inflammatory reaction that is usually caused by a virus infection but sometimes by poisoning (intoxication). Long treatment with prednisolone or azathioprine is usually successful, but early recognition of the disease increases the likelihood of success. Nowadays, chronic hepatitis due to hepatic copper accumulation in Beddlington terriers can be detected by DNA tests. Such tests make it possible to distinguish between carriers and non-carriers. Affected animals can be kept symptom-free by life-long treatment with zinc gluconate or penicillamine.
A serologic survey for leptospirosis in stray dogs from Moreno, Province of Buenos Aires, was carried out. Sera from 143 randomly collected animals were examined by the microscopic agglutination test. A total of 73 (51.0%) of the dogs have had contact with leptospirae. The predominant leptospiral agglutinnins were to serovar canicola (37.0%) and to a lesser degree against the serovar antigens pyrogenes (4.8%) and autumnalis (2.0%). Although a high percentage of seropositive reactions occurred against the serovar antigens ballum, pyrogenes and icterohaemorrhagiae, all the reactions against ballum and icterohaemorrhagiae, and the majority of the reactions to pyrogenes represented the natural coagglutinins frequently associated with serovar canicola. The detection of antibodies to the Autumnalis group in dogs of Argentina could be new and suggest the emergence of canine infections caused by a serovar of this serogroup which should be sought out and identified.