Vertical transmission of Anaplasma platys and Leishmania infantum in dogs during the first half of gestation.

BACKGROUND: Leishmania infantum is a canine zoonotic vector-borne protozoan pathogen transmitted by phlebotomine sand flies, whereas Anaplasma platys is a bacterium most likely transmitted by ticks. While vertical transmission of L. infantum from pregnant bitches to their offspring has been documented, thus far no studies have explored the possibility of vertical transmission of A. platys in dogs. This study investigated the occurrence of vertical transmission of L. infantum and A. platys in sheltered dogs during the first half of gestation, in an area of southern Italy characterised by a high incidence of infection by both pathogens. METHODS: The study population included 20 bitches (n = 10 pregnant, at 25-35 days of pregnancy; n = 10 non-pregnant), all subjected to ovariohysterectomy, which were examined for the presence of L. infantum and A. platys via cytological screening of bone marrow and whole blood samples. Infection by L. infantum and A. platys was also tested by immunofluorescence antibody test (IFAT) and quantitative real-time PCR (qPCR) targeting both pathogens. Selected tissue samples (n = 210) collected during surgical procedures from bitches and foetuses (n = 20) were assessed for the presence of L. infantum and A. platys by qPCR targeting a fragment of the kinetoplast minicircle DNA (kDNA) and the 16S rRNA gene, respectively. RESULTS: Leishmania infantum DNA was not amplified from either uteri or ovaries from pregnant bitches or foetal tissue samples, whereas a subset of ovarian (n = 2) and uterine (n = 4) tissue samples from non-pregnant bitches were infected, with parasite loads of up to 3.09 × 10 and 7.51 parasite/PCR reaction, respectively. Conversely, uterine (n = 10) and ovarian (n = 8) tissues from both pregnant and non-pregnant bitches, together with a subset (n = 5) of foetal tissue samples were qPCR positive for A. platys. Leishmania infantum and A. platys nucleic acids were amplified from two uteri from non-pregnant bitches, with parasite loads of up to 2.32 × 10(-3) and 2.05 parasite/per PCR reaction, respectively. CONCLUSIONS: Results from this study suggest that, in contrast to L. infantum, A. platys can be transmitted from pregnant dogs to their offspring during the first half of gestation. This hypothesis remains to be verified, for instance via direct observation of parasites in postpartum foetal tissues.

Evaluation of blood and bone marrow in selected canine vector-borne diseases.

BACKGROUND: Bone marrow (BM) is a major hematopoietic organ that can harbour a variety of vector-borne pathogens; however, knowledge of BM pathological changes in dogs infected with vector-borne pathogens is limited. Thus, the aim of the present study was to assess the pathological changes in canine BM associated with natural infections by four vector-borne pathogens, as well as to determine the relationships between such changes and abnormalities of the peripheral blood. METHODS: Cytological disorders and pathological changes of the BM of 83 dogs naturally-infected with one or more of four vector-borne pathogens (i.e., Anaplasma platys, Leishmania infantum, Babesia vogeli and Hepatozoon canis) were evaluated and compared with the corresponding hematological findings. RESULTS: Dysgranulopoiesis and dysmegakaryocytopoiesis were the most frequently observed BM abnormalities in infected dogs. Erythroid suppression, and lymphocytic, monocytic and macrophage hyperplasia were also observed. Interestingly, associations between suppression and hyperplasia of specific cell lines in the marrow and corresponding changes in numbers of circulating peripheral blood cells were not observed. CONCLUSIONS: Infections with one or more of the vector-borne pathogens examined in this study should be considered as differential diagnoses for secondary dysmyelopoiesis.

Anaplasma platys in bone marrow megakaryocytes of young dogs.

Anaplasma platys is an obligate intracellular rickettsial pathogen that infects platelets of dogs, forming basophilic intracellular morulae. In the present report, cellular inclusions were documented in bone marrow thrombocyte precursors of two young naturally infected dogs, indicating that A. platys can infect megakaryocytes and promegakaryocytes.

Failure of imidocarb dipropionate and toltrazuril/emodepside plus clindamycin in treating Hepatozoon canis infection.

Hepatozoonosis caused by Hepatozoon canis (Eucoccidiorida, Hepatozoidae) is among the most widespread vector-borne infections of dogs, primarily transmitted by Rhipicephalus sanguineus sensu lato ticks. Based on the absence of a consensus on the treatment regimes for canine hepatozoonosis, the present study aimed to evaluate the efficacy of imidocarb dipropionate (5-6 mg/kg subcutaneously once a week for 6 weeks), and of toltrazuril/emodepside (Procox(®), 15 mg/kg once a day for 6 days) in association with clindamycin (15 mg/kg once a day for 21 days) in treating naturally infected dogs. At the enrollment time (T0), 32 dogs, cytologically or molecularly positive for H. canis, were assigned to test and control groups. Animals were treated according to the specific therapeutic protocol, and the presence of H. canis gamonts was assessed weekly by cytology and PCR throughout six months (T1-T19). In addition, any abnormality in leucocyte morphology was evaluated and recorded. Results indicate that, in spite of a reduction in the percentage of infected dogs, both treatments did not provide parasitological cure. Accordingly, new treatment protocols or active compounds against H. canis should be investigated.

Efficacy of an imidacloprid/flumethrin collar against fleas, ticks and tick-borne pathogens in dogs.

BACKGROUND: Tick-borne diseases comprise a group of maladies that are of substantial medical and veterinary significance. A range of tick-borne pathogens, including diverse species of bacteria and protozoa, can infect both dogs and humans. Hence, the control of tick infestations is pivotal to decrease or prevent tick-borne pathogen transmission. Therefore, different commercial products with insecticidal, repellent or both properties have been developed for use on dogs. Recently, a collar containing a combination of imidacloprid 10% and flumethrin 4.5% has proven effective to prevent tick and flea infestations in dogs under field conditions and the infection by some vector-borne pathogens they transmit under laboratory-controlled conditions. METHODS: From March 2011 to April 2012, a field study was conducted in a private shelter in southern Italy to assess the efficacy of the imidacloprid/flumethrin collar against tick and flea infestations and to determine if this strategy would decrease tick-borne pathogen transmission in young dogs. A total of 122 animals were enrolled in the study and randomly assigned to group A (n = 64; collared) or group B (n = 58; untreated controls). Dogs were examined monthly for ticks and fleas and systematically tested for selected tick-borne pathogens. RESULTS: Compared to controls, the collar provided overall efficacies of 99.7% and 100% against tick and flea infestation, respectively. The overall efficacy for the prevention of tick-borne pathogens (i.e., Anaplasma platys and Babesia vogeli) was 91.6%. CONCLUSIONS: This study demonstrates that the imidacloprid/flumethrin collar is efficacious against flea and tick infestation as well as tick-borne pathogen transmission to dogs under field conditions.

Are vector-borne pathogen co-infections complicating the clinical presentation in dogs?

BACKGROUND: Infection by two or more canine vector-borne disease (CVBD)-causing pathogens is common in subtropical and tropical regions where vectors are plentiful. Co-infections may potentiate disease pathogenesis, thereby altering clinical manifestations typically associated with singular infections. These factors complicate diagnosis, treatment and can adversely influence prognosis if the practitioner fails to suspect, document, and treat each concurrent infection. The spectrum of pathogens co-infecting dogs may change over time in a given practice location due to the rapid expansion of arthropods and their associated vectored agents, and international transit among pets and wild animals. This applies, for example, to Dirofilaria immitis and Leishmania infantum, the distributions of which have expanded from northern to southern Italy, and vice versa, respectively. Indeed, mixed infections by D. immitis and L. infantum have only been reported once in Italy, probably due to the fact that competent vectors for these infections do not usually occur in the same geographical areas. Thus, information that would help practitioners to identify clinical presentations in dogs co-infected by D. immitis and L. infantum and other CVBD-causing pathogens is scant. FINDINGS: This manuscript describes the clinical history and physical examination of findings for 7 CVBD co-infected dogs that were examined because of a spectrum of clinical signs. Five dogs were co-infected with L. infantum and Ehrlichia canis, one dog with L. infantum, E. canis and D. immitis and the remaining dog with L. infantum and D. immitis. CONCLUSIONS: The clinical signs and haematological abnormalities associated with the diagnostic evaluation and treatment of these dogs is discussed. Also, the usefulness of bone marrow specimens for the molecular diagnosis of CVBDs and for the enhanced monitoring of treatment response is emphasized.

Prevention of canine leishmaniosis in a hyper-endemic area using a combination of 10% imidacloprid/4.5% flumethrin.

BACKGROUND: Dogs are the main reservoir hosts of Leishmania infantum, the agent of human zoonotic visceral leishmaniosis. This study investigated the efficacy of a polymer matrix collar containing a combination of 10% imidacloprid and 4.5% flumethrin as a novel prophylactic measure to prevent L. infantum infections in young dogs from a hyper-endemic area of southern Italy, with a view towards enhancing current control strategies against both human and canine leishmaniosis. METHODOLOGY/PRINCIPAL FINDINGS: The study was carried out on 124 young dogs, of which 63 were collared (Group A) while 61 were left untreated (Group B), from March-April 2011 until March 2012. Blood and skin samples were collected at baseline (April 2011) and at the first, second, third and fourth follow-up time points (July, September 2011 and November 2011, and March 2012, respectively). Bone marrow and conjunctiva were sampled at baseline and at the fourth follow-up. Serological, cytological and molecular tests were performed to detect the presence of L. infantum in the different tissues collected. At the end of the trial, no dog from Group A proved positive for L. infantum at any follow-up, whereas 22 dogs from Group B were infected (incidence density rate = 45.1%); therefore, the combination of 10% imidacloprid and 4.5% flumethrin was 100% efficacious for the prevention of L. infantum infection in young dogs prior to their first exposure to the parasite in a hyper-endemic area for CanL. CONCLUSIONS: The use of collars containing 10% imidacloprid and 4.5% flumethrin conferred long-term protection against infection by L. infantum to dogs located in a hyper-endemic area, thus representing a reliable and sustainable strategy to decrease the frequency and spread of this disease among the canine population which will ultimately result in the reduction of associated risks to human health.

Apparent tick paralysis by Rhipicephalus sanguineus (Acari: Ixodidae) in dogs.

Certain tick species including Ixodes holocyclus can inoculate neurotoxins that induce a rapid, ascending flaccid paralysis in animals. Rhipicephalus sanguineus, the most widespread tick of dogs, is recognized as a vector of several pathogens causing diseases in dogs and humans. A single report suggests its role as cause of paralysis in dogs. This study presents the clinical history of 14 young dogs heavily infested by R. sanguineus (intensity of infestation, 63-328) in an endemic area of southern Italy. During May to June of 2011, dogs were presented at the clinical examination with neurological signs of different degrees (e.g., hind limb ataxia, generalized lethargy, and difficulty in movements). All animals were treated with acaricides and by manual tick removal but ten of them died within a day, displaying neurological signs. The other 4 dogs recovered within 3 days with acaricidal and supportive treatment. Twelve dogs were positive by blood smear examination for Hepatozoon canis with a high parasitemia, two also for Babesia vogeli and two were negative for hemoparasites. Low-grade thrombocytopenia, hypoalbuminemia, and pancytopenia were the haematological alterations most frequently recorded. Other causes of neurological disease in dogs were excluded and the diagnosis of tick paralysis by R. sanguineus was confirmed (ex juvantibus) by early and complete recovery of 4 dogs following acaricidal treatment and tick removal.

Evolution of clinical, haematological and biochemical findings in young dogs naturally infected by vector-borne pathogens.

Longitudinal studies evaluating the evolution of clinical, haematological, biochemical findings in young dogs exposed for the first time to multiple vector-borne pathogens have not been reported. With the objective of assessing the evolution of clinical, haematological and biochemical findings, these parameters were serially monitored in naturally infected dogs throughout a 1-year follow-up period. Young dogs, infected by vector-borne pathogens based on cytology or polymerase chain reaction, were examined clinically and blood samples were obtained at seven different follow-up time points. Dogs were randomized to group A (17 dogs treated with a spot-on formulation of imidacloprid 10% and permethrin 50%) or to group B (17 dogs untreated). In addition, 10 4-month-old beagles were enrolled in each group and used as sentinel dogs. At baseline, Anaplasma platys was the most frequently detected pathogen, followed by Babesia vogeli, Bartonella spp., Ehrlichia canis and Hepatozoon canis. Co-infections with A. platys and B. vogeli, followed by E. canis and B. vogeli, A. platys and H. canis and A. platys and Bartonella spp. were also diagnosed. In dogs from group B, abnormal clinical signs were recorded at different time points throughout the study. No abnormal clinical signs were recorded in group A dogs. Thrombocytopenia was the most frequent haematological alteration recorded in A. platys-infected dogs, B. vogeli-infected dogs and in dogs co-infected with A. platys and B. vogeli or A. platys and Bartonella spp. Lymphocytosis was frequently detected among dogs infected with B. vogeli or co-infected with A. platys and B. vogeli. Beagles were often infected with a single pathogen rather than with multiple canine vector-borne pathogens. There was a significant association (p<0.01) between tick infestation and A. platys or B. vogeli, as single infections, and A. platys and B. vogeli or A. platys and Bartonella spp. co-infections. This study emphasizes the clinical difficulties associated with assigning a specific clinical sign or haematological abnormality to a particular canine vector-borne disease.

Diagnosis of canine vector-borne diseases in young dogs: a longitudinal study.

Canine vector-borne diseases (CVBDs) pose a diagnostic challenge, particularly when a dog is coinfected with more than one pathogen. The purpose of this study was to generate information about the diagnosis of CVBDs in young dogs following their first exposure to flea, tick, sand fly, louse, and mosquito vectors. From March 2008 to May 2009, 10 purpose-bred young naive beagle dogs and a cohort of 48 mixed-breed dogs living in an area to which CVBD is endemic in southern Italy were monitored using different diagnostic tests (cytology, serology, and PCR). Overall, PCR detected the highest number of dogs infected with Anaplasma platys, Babesia vogeli, and Ehrlichia canis, whereas seroconversion was a more sensitive indicator of exposure to Leishmania infantum. For A. platys infection, combining blood and buffy coat cytology in parallel enhanced the relative sensitivity (SE(rel)) (87.3%). For B. vogeli, the best diagnostic combination was buffy coat cytology and serology used in parallel (SE(rel), 67.5%), whereas serology and PCR used in parallel (SE(rel), 100%) was the best combination for L. infantum. Overall, 12 (20.7%) dogs were coinfected; however, the percentage of new coinfections decreased from baseline (50%) to the first (33.3%) and second (16.6%) follow-up time points. Numbers of coinfections with A. platys and B. vogeli were significantly higher (P < 0.05) than coinfections with other pathogen combinations. The data generated in this study provide insights on the incidence of certain pathogens infecting young dogs in southern Italy, highlight important diagnostic testing limitations, and support the use of multiple diagnostic modalities when attempting to confirm a tick-borne infection in an individual dog or in a canine population.

Clinical and laboratory monitoring of dogs naturally infected by Leishmania infantum.

The clinical evolution of Leishmania infantum infection in dogs is largely influenced by the host's individual immune response. Few studies have investigated the time course and clinical evolution of the infection both under experimental and natural conditions. In the present investigation, the time course of L. infantum infection was studied by monitoring clinical and laboratory features in naturally infected dogs sheltered in southern Italy. Twenty-three dogs that had one or more positive diagnostic tests for L. infantum were enrolled in the study and followed up every 4 months. A clinical score was assigned at each visit after assessing the presence of clinical signs suggestive of leishmaniosis. L. infantum-infected dogs were classified into three different categories based on their clinical score and serological and parasitological test results. Based on data from diagnostic tests and clinical scores, the time course of infection was defined as transient asymptomatic infection (11 dogs), persistent asymptomatic infection (2 dogs), and symptomatic infection (8 dogs). Two dogs were lost after the first sand fly season. The results of the present study provide a framework for assessing the clinical status of L. infantum infection in dogs and suggest that infected animals should be monitored over time to expedite therapeutic decisions and plan appropriate control interventions.

Monoclonal gammopathy associated with heartworm disease in a dog.

A 12-year-old, intact female, mixed Yorkshire terrier was evaluated for syncopal episodes, weakness, decreased appetite, and weight loss. Heartworm disease was diagnosed based on evidence of circulating microfilariae of Dirofilaria immitis on direct examination of blood smears and a positive SNAP heartworm antigen test. An immunoglobulin G (IgG) gammopathy, demonstrated by serum protein electrophoresis, was associated with heartworm disease in this dog. Response to treatment with both an adulticide and the microfilaricide ivermectin included remission of clinical signs and a decrease in the monoclonal gammopathy. To our knowledge, this is the first report of an IgG gammopathy associated with heartworm disease in the dog.

Canine leishmaniasis in Southern Italy: a role for nitric oxide released from activated macrophages in asymptomatic infection?

BACKGROUND: Human and canine leishmaniasis (CanL) by Leishmania infantum is endemic in Italy, with a high percentage of infected asymptomatic animals. However, the immune response mechanisms underlying the clinical presentation of CanL have not been fully investigated. Among leishmanicidal molecules produced by activated macrophages, nitric oxide (NO) produced by an inducible NO synthase seems to play an important protective role, but no conclusive data are available. Therefore, NO released by cultured macrophages from dogs with natural Leishmania infection living in an endemic area for CanL was evaluated. METHODS: On the basis of one year's clinical and laboratory follow-up, 22 dogs infected by Leishmania infantum were identified and grouped as: asymptomatic dogs (n = 13) and dogs with symptoms of leishmaniasis (n = 9). Each animal was bled twice at 4-month intervals and macrophage and lymphocyte cultures were obtained from peripheral blood mononuclear cells. Supernatants of L. infantum-infected macrophage cultures, with or without addition of autologous lymphocytes, were assayed for NO production by Griess reaction for nitrites. RESULTS: In the first months of the infection the levels of NO in supernatants of Leishmania-infected macrophages were higher in symptomatic than in asymptomatic dogs, but they were significantly increased in the latter group eight months after the diagnosis of infection. Furthermore, NO release significantly decreased in the presence of autologous lymphocytes in both groups of animals. CONCLUSION: These results suggest that NO may be involved in the long-term protection of dogs against natural Leishmania infection and in the clinical presentation of canine leishmaniasis in the Mediterranean area.

Application of 10% imidacloprid/50% permethrin to prevent Ehrlichia canis exposure in dogs under natural conditions.

Canine monocytic ehrlichiosis (CME) caused by Ehrlichia canis is the most known canine tick-borne disease (TBD) spread throughout the world. Preventing tick bites is a priority to reduce the risk of TBDs and it was the aim of the present study to evaluate the efficacy of a combination of imidacloprid 10% and permethrin 50% (ImPer) (Advantix; Bayer AG, Germany) in a spot-on formulation to control CME under field conditions. On January-March 2005, 845 dogs from two kennels in southern Italy (kennels of Bari (KB)- and Ginosa (KG)), with a history of tick infestation were initially tested by serology and PCR assay for E. canis infection. Data on Leishmania infantum infection were also available from a previous study carried out on the same dog population. One hundred twenty-six dogs (14.9%) presented anti-E. canis antibodies with a relative prevalence of 15.6% (n=65 dogs in KB) and 14.2% (n=61 dogs in KG). Five hundred thirty-five animals found negative both for E. canis and L. infantum infections were enrolled in three groups (Group A--treated with ImPer once a month; Group B--treated every 2 weeks; and Group C--untreated control animals) and monitored for E. canis infection by serology and PCR in November 2005 (first follow-up) and in March 2006 (second follow-up). The E. canis infection was serologically revealed, at the first and/or second follow-up, in 26 animals from Group C in KB and KG (mean incidence density rate (IDR), 13.24%) while in none of the animals from Group A (KB and KG) and only in one animal from Group B (IDR 1.13%) in KG. The final protection efficacy of ImPer ranged from 95.57% to 100% in Groups B and A. At PCR only 15 dogs from KG were positive for Rickettsiales only at the first follow-up and at the sequence analysis two (both in Group C) revealed 100% homology with E. canis sequences while 13 with Anaplasma platys. Four out of 13 A. platys PCR-positive dogs were also seropositive for E. canis at one or both follow-ups. ImPer, by virtue of its repellent and acaricidal activity against ticks, has been shown to be efficacious to prevent E. canis infection in treated dogs living under natural conditions in endemic areas.

Lymphocutaneous and nasal sporotrichosis in a dog from southern Italy: case report.

Sporotrichosis is a chronic, granulomatous and usually lymphocutaneous infection of humans and animals caused by the dimorphic fungus, Sporothrix schenckii. This study reports a case of lymphocutaneous and nasal sporotrichosis in a hunting dog with a three month history of non-healing skin lesions. Cytological examination of nasal discharge and of the material collected from ulcerated skin surfaces showed a few cigar-shaped organisms within macrophages. Fungal cultures of nasal and ulcerated skin swabs yielded colonies of S. schenckii. The dog received oral itraconazole but died of unrelated causes. Necropsic examination was not performed.

Recombinant K39 dipstick immunochromatographic test: a new tool for the serodiagnosis of canine leishmaniasis.

The spread of human leishmaniasis has prompted the scientific community to study dogs as reservoirs for Leishmania infantum. Canine leishmaniasis (CanL) is widespread in the Mediterranean area with a prevalence of up to 50%. The first step toward controlling the disease is to monitor its distribution, mainly in stray dogs. The validity of a recombinant K39 (rK39) dipstick test, commercially available for the serodiagnosis of human leishmaniasis, was evaluated using sera from 165 dogs selected on the basis of positive or negative lymph node smears at parasitological examination. The results were compared with the indirect fluorescent antibody test (IFAT) (cutoff 1:80). Sera from a group of dogs with other diagnosed diseases but negative for leishmaniasis were also tested to evaluate any cross-reactivity. Various procedures were used for testing whole blood samples. The relative specificity of the rK39 dipstick and IFAT was 100% (97 of 97) and 98.97% (96 of 97), whereas the relative sensitivity was 97.06% (66 of 68) and 98.53% (67 of 68), respectively. The results of the dipstick and IFAT corresponded except for 2 sera (k = 0.987). This data confirm the usefulness of rK39 antigen for diagnosing CanL both in symptomatic and asymptomatic dogs. The rK39 dipstick proved to be a rapid, sensitive, and specific test that may be very useful in the field for large-scale screening and also in veterinary practice, requiring minimal equipment and operator expertise.

Otodectes cynotis (Acari: Psoroptidae): examination of survival off-the-host under natural and laboratory conditions.

The biological and environmental factors affecting survival off-the-host of Otodectes cynotis (Acari: Psoroptidae) ear mites were investigated under natural and laboratory conditions. From November 2000 to November 2002 mites were collected monthly from cats and divided into four groups according to sex and stage. In laboratory conditions, the mites were placed in an incubator with a steady 95% relative humidity (r.h.), a 10 degrees C. All the plates were examined by stereomicroscopy every 24 h until all the mites had died. The data were analysed statistically by multiple linear regression and survival analysis. At 10 degrees C, the maximum survival time of mites was between 15 and 17 days, while at 34 degrees C, it was between 5 and 6 days. The maximum survival time of adult females was significantly longer than that of other stages. No differences were observed in maximum survival times of mites that had been offered food and those that had not, or in the time (in days) to reach 50% mortality (LT50). When exposed to environmental conditions, the maximum survival time (12 days) was observed at temperatures ranging from 12.3 to 14.2 degrees C and r.h.s between 57.6 and 82.9%. Multiple regression analysis showed that temperature alone influenced the maximum survival time and LT50 of mites, and that the rate of survival declined linearly with increasing mean temperature. This basic understanding of off-host survival suggests that, places which have been inhabited by infected animals may need to be disinfected or remain vacated for at least 12 days before occupancy by clean cats or dogs.