Tommaso Furlanello

Babesia canis canis and Babesia canis vogeli clinicopathological findings and DNA detection by means of PCR-RFLP in blood from Italian dogs suspected of tick-borne disease.

The aims of this study were to determine the presence of Babesia spp. in blood samples from Italian dogs with clinical signs compatible with tick-borne diseases by means of PCR-restriction fragment length polymorphism (RFLP) and describe the clinicopathological findings of dogs with Babesia infection. We evaluated the majority of canine babesiosis cases by means of clinical history, physical examination, hematological, biochemical, serum electrophoresis, urinalysis and hemostatic tests. Forty-five out of 164 canine blood samples studied were positive to Babesia PCR-RFLP with the following results: Babesia canis canis (n=34) and Babesia canis vogeli (n=11). The majority of B. c. canis infections were detected in Northern Italy (29.1%; 30/103). B. c. vogeli cases were detected mainly in Central and Southern Italy (16.3%; 10/61). Only one B. c. vogeli was detected in Northern Italy (0.9%; 1/103). Three positive samples to B. c. canis and four positive samples to B. c. vogeli were selected for sequencing of a fragment of the 18S rRNA gene (410bp) for further molecular characterization. The sequence obtained from all seven dogs was 99/100% homologous to sequences from B. c. canis and B. c. vogeli, respectively, present in GenBank. Sixty-two percent of dogs infected with B. c. canis had recently travelled on a hunting trip to East European countries. The main acute clinical signs were dehydration, apathy, anorexia and fever. The majority of dogs infected with B. c. canis presented at initial clinical examination mild to severe thrombocytopenia, hyperfibrinogenemia, mild to moderate normocytic-normochromic non-regenerative anemia, hemolysis and neutropenia. The urinalysis showed hemoglobinuria in 13/19 dogs suggesting intravascular hemolysis. Dogs with B. c. canis infection had high levels of C-reactive protein. Hypoalbuminemia was present in 17/26 dogs. The 11 cases of B. c. vogeli infection did not present a homogenous clinicopathological pattern. B. c. vogeli infections were observed in young dogs causing hemolytic anemia and in adult/old does that frequently presented predisposing factors such as splenectomy or immunocompromised conditions. In conclusion, this study demonstrates the presence of B. c. canis and B. c. vogeli in Italian sick dogs and differences in clinicopathological pattern in these two species of B. canis.

Hematologic abnormalities and flow cytometric immunophenotyping results in dogs with hematopoietic neoplasia: 210 cases (2002-2006).

BACKGROUND Growing interest in veterinary oncohematology has facilitated the recent development and advancement of new techniques, such as flow cytometry, for immunophenotyping hematopoietic neoplasia in animals. OBJECTIVE The aim of this retrospective study was to characterize hematologic abnormalities and flow cytometric immunophenotyping (FCI) results in cases of hematopoietic neoplasia in dogs. METHODS Signalment, CBC data, and FCI results were obtained for 210 dogs with blood samples submitted to our laboratory. Immunophenotyping was carried out using an Epics XL-MCL flow cytometer and a panel of 10 antibodies (CD45, CD3, CD4, CD8, CD79, CD21, CD14, CD34, CD41/61, CD61). The prevalence and severity of hematologic abnormalities was determined for the different types of hematopoietic neoplasms. RESULTS Based on cell morphology and phenotype, cases were classified as: acute lymphoblastic leukemia (ALL, n=51), acute myeloid leukemia (AML, n=33), chronic lymphocytic leukemia (CLL, n=61), and leukemic high-grade lymphoma (L-HGL, n=65). Most cases of ALL (47/51) and L-HGL (41/65) had a B-cell phenotype, while most cases of CLL (54/61) had a T-cell phenotype, with a high prevalence of the large granular lymphocyte subtype (49/61). Anemia was found in 85% of all cases and was significantly more severe in ALL and AML compared with CLL and L-HGL. Neutropenia was seen in 64-78% of acute leukemias (AML and ALL) in contrast to no cases of CLL and 11% of L-HGL. Thrombocytopenia was seen in 88-90% of acute leukemias in contrast to 15% of CLL and 40% of L-HGL. Thrombocytopenia was more prevalent (71% vs 22%) and significantly more severe in T-cell vs B-cell L-HGL. CONCLUSION A standard CBC is useful in suggesting the type of hemoproliferative disorder and may also help to predict the phenotype, especially in cases of L-HGL.

Febrile Illness Associated with Rickettsia conorii Infection in Dogs from Sicily

We report serologic and molecular evidence of acute, febrile illness associated with Rickettsia conorii in 3 male Yorkshire terriers from Sicily (Italy).

A serological and molecular study of Leishmania infantum infection in cats from the Island of Ibiza (Spain).

The aim of this study was to evaluate the prevalence of Leishmania infantum infection within a feline population by serologic and molecular methods and to identify associated risk factors. One hundred five cats living outdoors were studied. Sera were tested for IgG antibodies against L. infantum, Toxoplasma gondii, and feline immunodeficiency virus (FIV) and for the detection of feline leukemia virus (FeLV) p27 antigen by enzyme-linked immunosorbent assay (ELISA). L. infantum real-time polymerase chain reaction (PCR) was performed on DNA extracted from blood. L. infantum and T. gondii seroprevalence rates were 13.2% and 55.2%, respectively. The prevalence of L. infantum by PCR was 8.7%. The total rate of L. infantum infection derived from seroreactivity and/or positive PCR was 15.4%. Serology and PCR results were positively associated, and moderate agreement (kappa = 0.489) was found between Leishmania ELISA and PCR. No statistical association was found between positive Leishmania PCR results and gender, clinical status, or T. gondii seropositivity. Six of the 105 cats (5.7%) displayed clinical signs compatible with feline cutaneous leishmaniosis, and 4 out of these 6 cats (66.7%) were found to have Leishmania infection by means of serology and/or PCR. Leishmania seropositivity was associated with clinical signs of feline cutaneous leishmaniosis (p = 0.029). The prevalence of FeLV p27 antigen was 16.2% (17/105) and of FIV antibody was 20.9% (22/105), with coinfection found in 9.5% (10/105) of the cats. Leishmania ELISA seroreactivity and positive PCR results were statistically associated with FeLV infection and with coinfection of both retroviruses but not with a positive FIV status. The high seroprevalence and molecular rates of Leishmania infection observed indicate that cats are frequently infected with L. infantum, and the association with FeLV suggests a potential role for this retrovirus in feline Leishmania infection in endemic areas.

Use of Fine Needle Aspirates and Flow Cytometry for the Diagnosis, Classification, and Immunophenotyping of Canine Lymphomas:

Fifty canine lymphomas were classified cytomorphologically using the updated Kiel classification scheme. Aspirates of lymph nodes from dogs with lymphoma were stained using 5 canine-specific antibodies and 3 human-specific antibodies that cross-react with canine lymphocytes. The antibody-stained aspirates were analyzed by flow cytometry. A total of 32 (64%) of the 50 lymphomas were characterized as B-cell origin and 18 (36%) were of T-cell origin. B-cell lymphomas were identified in 12 females and 20 males with a mean age of 8.35 years. T-cell lymphomas were identified in 8 females and 10 males with a mean age of 7.9 years. A minority of the lymphomas were low-grade B-cell and T-cell lymphomas (6/50, 12% and 4/50, 8%, respectively). The most common morphologic types were high-grade centroblastic and unclassifiable plasmacytoid for B- and T-cell lymphomas (18/50, 36% and 7/50, 14%, respectively).

Serological diagnosis of canine leishmaniosis: comparison of three commercial ELISA tests (Leiscan®, ID Screen® and Leishmania 96®), a rapid test (Speed Leish K®) and an in-house IFAT

BackgroundSpeed Leish K® is used as a serological screening test for Leishmania infection prior to vaccination. Limited comparative serological studies with Speed Leish K® have been performed. The aim of this study was to evaluate the diagnostic performance of four commercially available serologic tests including ELISAs (Leiscan®, ID Screen® and Leishmania 96®), a rapid test (Speed Leish K®) and an in-house IFAT for the detection of specific antibodies against Leishmania infantum antigen in dogs in different states of infection.MethodsSick infected dogs (n = 36), healthy infected dogs (n = 18), L. infantum seropositive dogs with low to high levels of antibodies (n = 53), dogs seropositive to other pathogens (to evaluate cross reaction) (n = 14) and uninfected dogs from a non-endemic area (n = 50) and from an endemic area (n = 32) were analysed by the serological methods mentioned above.ResultsThe sensitivity was as follows: ID Screen® (0.953), Leiscan® and Leishmania 96® (0.925), IFAT (0.869) and Speed Leish K® (0.636). The maximum specificity (1.000) was attained for all diagnostic tests except the Leishmania 96® (0.896) and IFAT (0.917). The accuracy was as follows: ID Screen® (0.975), Leiscan® (0.961), Leishmania 96® (0.911), IFAT (0.892) and Speed Leish K® (0.808). In relation to the area under the ROC curve (AUC-ROC), the maximum value was attained with the ID Screen® (0.993) closely followed by Leiscan® (0.990), then, Leishmania 96® (0.962), IFAT (0.926) and Speed Leish K® (0.818). For the Kappa index, the best result was obtained by the ID Screen® (0.951) followed by Leiscan® (0.921), Leishmania 96® (0.822), IFAT (0.783) and Speed Leish K® (0.622). Statistically significant differences were found between the AUC-ROC of quantitative serological tests and the only qualitative rapid test evaluated. There were also statistically significant differences between AUC-ROC of the ELISAs (ID Screen® and Leiscan®) and IFAT.ConclusionsLeiscan® and ID Screen® had superior diagnostic performance measures than IFAT and all quantitative serological tests were superior when compared to Speed Leish K®. Thus, Speed Leish K® may be considered a less valuable screening test prior to vaccination as it may result in vaccination of seropositive dogs and in some cases seropositive sick dogs.

Serum acute phase protein concentrations in dogs with hyperadrenocorticism with and without concurrent inflammatory conditions.

BACKGROUND Acute phase proteins (APPs) are promising markers of inflammation in dogs, because they are more sensitive than WBC counts in detecting clinical and subclinical inflammation. Endogenous corticosteroids can mask an acute phase response and make it more difficult to identify underlying inflammatory disease. OBJECTIVE The purpose of this study was to evaluate the acute phase protein response in dogs with spontaneous hyperadrenocorticism (HAC) with and without concurrent inflammatory conditions. METHODS Serum concentrations of C-reactive protein (CRP), haptoglobin (Hp), fibrinogen, and albumin were measured in 44 healthy adult dogs and 39 dogs with HAC; the HAC group was further divided into dogs with and without concurrent infection/inflammation. A fourth group of dogs with severe sepsis and without HAC was compared with the dogs with HAC and severe sepsis. RESULTS Dogs with uncomplicated HAC had significantly higher Hp and fibrinogen concentrations compared with healthy control dogs (P<.001). Dogs with HAC and severe inflammatory disease also had significantly higher CRP and lower albumin concentrations than control dogs and dogs with HAC without concurrent inflammation. Dogs with sepsis but without HAC had significantly higher CRP concentrations than dogs with HAC and sepsis. CONCLUSIONS Dogs with HAC had increases in the moderate APPs (Hp and fibrinogen), and no significant changes in CRP and albumin compared with healthy dogs. Although concurrent HAC appeared to blunt the CRP response in dogs with sepsis, increased serum CRP concentration in dogs with HAC is likely indicative of severe concurrent inflammation.

Cytauxzoon sp infection in the first endemic focus described in domestic cats in Europe

Information about epidemiological and clinicopathological aspects of domestic cat infection by species of Cytauxzoon other than Cytauxzoon felis is limited and it has rarely been reported. Following the detection of clinical cytauxzoonosis in three cats from Trieste (Italy), an epidemiological study was carried out in colony (n=63) and owned (n=52) cats from the same city to investigate the presence of Cytauxzoon sp. infection and to assess clinicopathological findings and variables associated with this infection. Cytauxzoon sp. infection was detected by 18S rRNA gene PCR in 23% (27/118) and by blood smear examination in 15% (18/118) of domestic cats. The 18S rRNA gene sequences obtained were 99% identical to the Cytauxzoon sp. sequences deposited in GenBank(®) from Spanish, French and Mongolian wild and domestic cats. Erythroparasitemia was observed mainly in apparently healthy cats. Cytauxzoon sp. infection was statistically associated with the colony group and the outdoor life style. No statistical association was found between positivity by PCR and breed, gender, age, presence of ticks and/or fleas, clinical status, laboratory findings such as anemia, FIV and/or FeLV status and mortality rate. Persistence of the infection was monitored and documented in four clinical cases. We reported the first clinicopathological description of naturally occurring Cytauxzoon sp. infection in domestic cats living in Italy. The predominance of subclinical erythroparasitemia and the evidence of persistent infection support the hypothesis that the domestic cat might serve as a reservoir host for this infection.

Detection of Leishmania infantum DNA mainly in Rhipicephalus sanguineus male ticks removed from dogs living in endemic areas of canine leishmaniosis

BackgroundSand flies are the only biologically adapted vectors of Leishmania parasites, however, a possible role in the transmission of Leishmania has been proposed for other hematophagous ectoparasites such as ticks. In order to evaluate natural infection by Leishmania infantum in Rhipicephalus sanguineus ticks, taking into account its close association with dogs, 128 adult R. sanguineus ticks removed from 41 dogs living in endemic areas of canine leishmaniosis were studied.MethodsIndividual DNA extraction was performed from each tick and whole blood taken from dogs. Dog sera were tested for IgG antibodies to L. infantum antigen by ELISA and L. infantum real-time PCR was performed from canine whole blood samples and ticks.ResultsLeishmania infantum PCR was positive in 13 ticks (10.1%) including one female, (2.0%) and 12 males (15.2%), and in only five dogs (12.2%). Male ticks had a significantly higher infection rate when compared to female R. sanguineus. The percentage of L. infantum seroreactive dogs was 19.5%. All but two PCR positive dogs were seroreactive. Leishmania infantum PCR positive ticks were removed from seropositive and seronegative dogs with a variety of PCR results.ConclusionsThis study demonstrates high prevalence of L. infantum DNA in R. sanguineus ticks removed from L. infantum seropositive and seronegative dogs. The presence of L. infantum DNA was detected mainly in male ticks possibly due to their ability to move between canine hosts and feed on several canine hosts during the adult life stage. Additional studies are needed to further explore the role of R. sanguineus ticks and in particular, male adults, in both the epidemiology and immunology of L. infantum infection in dogs in endemic areas.

Detection of Leishmania infantum, Babesia canis, and rickettsiae in ticks removed from dogs living in Italy

The aims of this study were to determine natural infections by Anaplasma phagocytophilum/Anaplasma platys, Bartonella henselae, Ehrlichia canis, Leishmania infantum, Rickettsia spp., Babesia spp., and Hepatozoon spp. by molecular methods in ticks (n=91) removed from dogs with clinical signs and laboratory abnormalities compatible with tick-borne diseases (n=22) living in Italy and to assess the distribution and species of ticks encountered. Ticks from dogs living in southern Italy were all identified as Rhipicephalus sanguineus (n=25), ticks from central Italy included Rh. sanguineus (n=8) and Ixodes ricinus (n=9), ticks from northern Italy included Rh. sanguineus (n=45), Dermacentor marginatus (n=3), and one I. ricinus. Leishmania infantum, Rickettsia spp., and Babesia canis were the only pathogens detected in 7 (8%), 4 (4%), and 2 (2%) out of 91 ticks, respectively. L. infantum was detected in I. ricinus from central Italy and in Rh. sanguineus from northern and central Italy. Rickettsia conorii and Ri. massiliae were detected in Rh. sanguineus ticks from central and southern Italy (Sicily), respectively. Bab. canis was detected in D. marginatus ticks from northern Italy.