Manolis K. Chatzis

Cytological and molecular detection of Leishmania infantum in different tissues of clinically normal and sick cats

Natural infection of domestic cats by Leishmania infantum (synonym: L. chagasi) has been demonstrated in several European, Latin American, and Asian countries, and the estimated prevalence of infection, based mainly on blood PCR, ranges from 0.3% up to 60.6%. In this study we aimed to: (a) estimate the prevalence of the infection by L. infantum in clinically normal cats (group A; n=50) and in cats with various clinical signs (group B; n=50), living in an endemic region, by both cytological examination of four different tissues (lymph node, skin, bone marrow, and conjunctiva) and by PCR in four different tissues (blood, skin biopsies, bone marrow, and conjunctiva); (b) compare the diagnostic sensitivity of the above methods and evaluate for possible associations between their results; and (c) investigate the possible associations between infection by L. infantum and signalment, living conditions, season of sampling, and health status of the cats. The prevalence of the infection in the study population was 41% and did not differ (P=0.839) between group A (42%) and B (40%) cats. Lymph node, skin, bone marrow and conjunctiva cytology was always negative. Therefore, the diagnosis of the infection was based only on PCR in blood, skin biopsy, bone marrow and conjunctiva, which was positive in 13%, 18.2%, 16% and 3.1% of the cats, respectively. PCR was positive in only one of the four tissues in 80.5% of the infected cats. The results differed (P=0.014) among the four tissues and were less frequently positive in conjunctiva compared to skin biopsies and bone marrow (P=0.007 for both comparisons), thus highlighting the need for multiple tissue PCR testing in order to minimize false-negative results. More PCR-positive cats were found when sampling was performed during the period of sandfly activity (odds ratio: 2.44; P=0.022). Also, in group B cats, the likelihood of PCR-positivity was higher (odds ratio: 3.93; P=0.042) among those presenting at least one systemic clinical sign that had been previously reported in cats with leishmaniosis.

Evaluation of indirect immunofluorescence antibody test and enzyme-linked immunosorbent assay for the diagnosis of infection by Leishmania infantum in clinically normal and sick cats.

Abstract Cats that live in areas where canine and human leishmaniosis due to Leishmania infantum is endemic may become infected and may develop anti-Leishmania antibodies. In this study 50 clinically normal and 50 cats with cutaneous and/or systemic signs that lived in an endemic area and had been previously examined for infection by L. infantum using PCR in four different tissues were serologically tested for the presence of anti-Leishmania IgG (IFAT and ELISA) and IgM (IFAT). The aim was to compare the results of IFAT, ELISA and PCR and to investigate the possible associations between seropositivity to Leishmania spp and signalment, living conditions, season of sampling, health status of the cats, and seropositivity to other infectious agents. Low concentrations of anti-Leishmania IgG were detected by IFAT in 10% of the cats and by ELISA in 1%, whereas anti-Leishmania IgM were detected by IFAT in 1%. There was disagreement between the results of IFAT and ELISA for anti-Leishmania IgG (P = 0.039) and between all serological tests and PCR (P < 0.001). The diagnostic sensitivity of all serological tests, using PCR as the gold standard, was very low, but ELISA and IFAT for anti-Leishmania IgM had 100% specificity. The diagnostic sensitivity of all serological tests could not be improved by changing the cut-off values. Seropositivity for Leishmania spp was not associated with signalment, living conditions, season of sampling and health status of the cats or with seropositivity to feline leukemia virus, feline immunodeficiency virus, feline coronavirus, Toxoplasma gondii and Bartonella henselae. In conclusion, because of their low sensitivity and very high specificity two of the evaluated serological tests (ELISA for anti-Leishmania IgG and IFAT for anti-Leishmania IgM) may be useless as population screening tests but valuable for diagnosing feline infection by L. infantum.

Thyroid function in 36 dogs with leishmaniosis due to Leishmania infantum before and during treatment with allopurinol with or without meglumine antimonate

Hypothyroidism may predispose to the development of canine leishmaniosis or it may appear during the course of the latter due to infiltration and destruction of the thyroid gland by infected macrophages. The main purpose of this study was to evaluate thyroid function through measurement of serum total thyroxin (tT₄), free thyroxin (fT₄), and canine thyroid stimulating hormone (cTSH) concentrations in 36 dogs with leishmaniosis, before and after 2 and 4 weeks of treatment with allopurinol with or without meglumine antimonate. Before treatment 27/36 (75%) dogs had serum tT₄ concentrations below the lower limit of the reference interval but only 2 of them had concurrently serum fT₄ concentrations below the lower limit of the reference interval and none had increased serum cTSH concentrations. During treatment there were no significant changes in serum tT₄ or fT₄ concentrations, whereas a significant increase in serum cTSH was observed. Two dogs had decreased serum tT₄ and fT₄ but normal cTSH concentrations before treatment and two other dogs had decreased serum tT₄ and increased cTSH, but normal fT₄ concentrations during the treatment period. Although hypothyroidism could not be definitively excluded in these dogs it is considered unlikely based on their overall hormonal profile, clinical presentation, and response to treatment. Therefore, hypothyroidism does not appear to be an important predisposing disease or a frequent complication of canine leishmaniosis.

Comparison of two commercial rapid in-clinic serological tests for detection of antibodies against Leishmania spp. in dogs

Antibodies against Leishmania spp. are detected in most dogs with clinical signs of leishmaniasis due to Leishmania infantum. Accurate, rapid in-clinic serological tests may permit immediate confirmation of the diagnosis and implementation of therapeutic measures. The aim of the current study was to evaluate the diagnostic accuracy of 2 commercial, rapid in-clinic serological tests for the detection of anti-Leishmania antibodies in sera of dogs, the Snap Canine Leishmania Antibody Test kit (IDEXX Laboratories Inc., Westbrook, Maine) and the ImmunoRun Antibody Detection kit (Biogal Galed Labs, Kibbutz Galed, Israel), using indirect fluorescent antibody test (IFAT) as the reference method. A total of 109 sera collected from 65 seropositive and 44 seronegative dogs were used. The sensitivities of the Snap and ImmunoRun kits were 89.23% (95% confidence interval: 79.05–95.54%) and 86.15% (95% confidence interval: 75.33–93.45%), respectively, and the specificity of both tests was 100%. A good agreement between each of the rapid in-clinic serological tests and IFAT and between the 2 rapid in-clinic serological tests was witnessed. Both rapid in-clinic serological tests showed an adequate diagnostic accuracy and can be used for the fast detection of antibodies against L. infantum in dogs.

Serum pharmacokinetics of clindamycin hydrochloride in normal dogs when administered at two dosage regimens.

The aim of this cross-over study was to compare clindamycin pharmacokinetics in the serum of clinically normal dogs when administered orally at two dosage regimens (5.5 mg/kg, twice daily, and 11 mg/kg, once daily), separated by a 1 week wash-out period. Serum samples were obtained from six clinically normal laboratory beagles before, 3, 6, 9 and 12 h after the first and fifth dose of clindamycin at 5.5 mg/kg, twice daily, and before, 3, 6, 9, 12, 18 and 24 h after the first and third dose at 11 mg/kg, once daily. Serum clindamycin concentrations were determined by reverse-phase liquid chromatography coupled with mass spectrometry. Results were analysed using Students paired t-test, at a 5% level of significance. Values of pharmacokinetic parameters that differed significantly between the two dosage regimens included the following: maximal concentration and area under the concentration-time curve were higher at 11 mg/kg, once daily, than at 5.5 mg/kg, twice daily; and, more importantly, the ratio of AUC(0-24) to the minimal inhibitory concentration (MIC) value of 0.5 μg/mL for a 24 h period (AUC(0-24)/MIC) was higher when clindamycin was administered at 11 than at 5.5 mg/kg, at least during the first day of drug administration. Therefore, a better pharmacokinetic profile may be expected when clindamycin is administered at 11 mg/kg, once daily, for the treatment of canine pyoderma caused by Staphylococcus pseudintermedius.

Prospective evaluation of serum pancreatic lipase immunoreactivity and troponin I concentrations in Leishmania infantum-infected dogs treated with meglumine antimonate☆

Canine leishmaniosis (CanL) caused by Leishmania infantum is an important zoonotic disease. One of the most commonly used drugs for the treatment of CanL is meglumine antimonate. Drugs of this class have been associated with pancreatitis and cardiotoxicity in humans infected with Leishmania spp. The aim of this study was to measure serum canine pancreatic lipase immunoreactivity (Spec cPL) and cardiac troponin I (cTnI) concentrations in dogs with leishmaniosis during treatment with meglumine antimonate, and to compare them with those of dogs with leishmaniosis not treated with this drug. A total of 30 non-uremic dogs with leishmaniosis, living in Greece, were prospectively enrolled into the study. Of the 30 dogs, 20 (Group A) were treated with a combination of meglumine antimonate (100mg/kg, SC, q24 h) and allopurinol (10mg/kg, PO, q12h) for 28 days, while 10 dogs (Group B) were treated with allopurinol alone (10mg/kg, PO, q12h) for 28 days. Blood samples were collected at timepoint 0 (before treatment) and at 14 and 28 days after the initiation of treatment. None of the dogs treated with meglumine antiomonate had a Spec cPL concentration suggestive of pancreatitis (≥ 400 μg/L) or clinical signs suggestive of pancreatitis at any of the timepoints. Similarly, none of the dogs treated with meglumine antiomonate had a serum cTnI concentration above the upper limit of the reference range (>0.5 ng/mL) or clinical evidence of cardiotoxicity at any of the 3 timepoints. In the present study, meglumine antimonate treatment in dogs with leishmaniosis did not result in clinical or laboratory evidence of either pancreatitis or cardiotoxicity.

Molecular detection of vector-borne pathogens in Greek cats

Infectious diseases have been increasingly recognized in cats worldwide. The objective of this study was the molecular investigation of the prevalence of selected pathogens in healthy and sick cats from Greece, a country highly endemic for several canine vector-borne pathogens. Blood and/or bone marrow samples from 50 clinically healthy and 50 sick adult (>1 year-old) cats were retrospectively examined for the amplification of Bartonella spp., haemoplasmas, Ehrlichia spp., Anaplasma spp., Babesia spp., and Cytauxzoon spp. DNA. Overall, 14.9% of the cats were found to be infected or co-infected by haemoplasmas, including Candidatus Mycoplasma haemominutum and M. haemofelis. In addition, 8.5% of the cats were infected by Bartonella henselae, Bartonella clarridgeiae or Bartonella koehlerae. In contrast, DNA of Ehrlichia spp., Anaplasma spp., Babesia spp. and Cytauxzoon spp. was not amplified from the blood or bone marrow of any cat. There was no significant difference in either haemoplasma or Bartonella infection rates when comparing healthy and sick cats. This study represents the first description of Bartonella koehlerae in Greek cats.

Concentrations of clindamycin hydrochloride in homogenates of normal dog skin when administered at two oral dosage regimens

Background: Clindamycin is frequently used for the treatment of bacterial pyoderma. Objective: To compare the pharmacokinetics of clindamycin in whole skin homogenates of normal dogs when administered orally at two dosage regimens (5.5 mg/kg BW, twice daily and 11 mg/kg BW, once daily). Animals and Methods: Skin biopsies were obtained from six laboratory beagles before, 3, 6 and 12 h after the first and the fifth dose of clindamycin at the former regimen, as well as before, 3, 6, 12 and 24 h after the first and third dose at the latter regimen. Tissue was homogenized and clindamycin concentrations were measured by reverse-phase high-performance liquid chromatography coupled with mass spectrometry. Results were analyzed using Students t-test at a level of significance of 0.05. Results: Maximal concentration and area under the concentration–time curve, but not their relevant dose-normalized values, were higher at the dosage regimen of 11 mg/kg BW, once daily than at 5.5 mg/kg BW twice daily. Conclusions: The pharmacokinetic profile of clindamycin is at least equal, if not better, when this antimicrobial is administered at 11 mg/kg BW, once daily.