Cercopithifilaria spp. of dogs: little known but prevalent filarioids beneath the skin.

Filarioids of the genus Cercopithifilaria are little studied, yet widespread parasites, that are relatively unique in being one of the very few nematodes transmitted by hard ticks. These filarioids live in the subcutis while microfilariae are found in the dermis. Definitive hosts include domestic dogs as well as a wide range of vertebrates, such as ruminants, non-human primates, murids, marsupials, porcupines, viverrids, bears and lagomorphs. The genus Cercopithifilaria contains three taxa (i.e. C. bainae, C. grassii and a yet undescribed species, namely Cercopithifilaria sp. II) that are known to infect dogs worldwide, with their occurrence overlapping the distribution of the main tick vector, Rhipicephalus sanguineus sensu lato. In recent decades, more attention has focused on these filarioids since they have been associated with clinical signs of infection, such as dermatitis, chronic polyarthritis and cutaneous cysts, and possibly with facilitating infections caused by other tick-borne pathogens. Nevertheless, these parasites remain largely underdiagnosed in clinical practice due to the lack of awareness of veterinary practitioners and to major obstacles to their diagnosis. In this review, we have assessed currently available data on Cercopithifilaria spp. infecting dogs worldwide and discussed the biological, clinical and epidemiological aspects of these filarioids, with the overall aim to gain a better understanding of their potential role in skin diseases.

Anisakis pegreffii Larvae in Sphyraena viridensis and Description of Granulomatous Lesions.

The aim of the present study was to describe gastric granuloma caused by Anisakis pegreffii in Sphyraena viridensis caught in the central Mediterranean Sea. Sixty-eight S. viridensis specimens were collected from different fish markets on the east coast of Sicily. Coelomic organs were observed both macroscopically and with the aid of stereomicroscope. Parasite specimens and lesioned tissues were collected for identification, histological and molecular analyses. Twelve specimens (p = 17.6%) were positive for the presence of nematode larvae, morphologically identified as larvae of Anisakis sp., with values of mean abundance and mean intensity of 0.9 and 4.8, respectively. One large female specimen showed massive parasite infection associated with nodular lesions of the gastric wall. By histology, several nematode larvae encysted through the gastric wall were found. The parasite bodies were surrounded by a granulomatous reaction made up of macrophages, epithelioid cells, some lymphocytes and an external connective sheet. Molecular analysis of 18S rRNA and cox2 genes from Anisakis sp. collected larvae, identified them as A. pegreffii. The lesions here described, though macroscopically superimposable on human eosinophilic granuloma, microscopically showed significant differences in the inflammatory cells involved and in the type of immune reaction.

Bovine besnoitiosis in a cattle herd in Sicily: an isolated outbreak or the acknowledgment of an endemicity?

Bovine besnoitiosis is a debilitating infectious disease caused by Besnoitia besnoiti (Apicomplexa; Sarcocystidae). The disease is mainly characterized by cutaneous and systemic signs, infertility in bulls, and abortion in cows. The current study describes an autochthonous outbreak of bovine besnoitiosis in Sicily, Southern Italy, being the first report of B. besnoiti infection in the island so far. In a cattle farm located in Syracuse province, a 4-year-old Belgian blue bull born in Sicily displayed typical clinical signs of the sub-acute/chronic disease phase with thickening of the skin of the scrotum and testicular degeneration. Histological examination of scrotal biopsies revealed the presence of several tissue cysts of B. besnoiti. The serological analysis of the herd using a commercial ELISA revealed a high seroprevalence (45 out of 55; 82%) of antibodies against B. besnoiti. Few seropositive animals (5 out of 45; 11%) showed clinical signs, cysts in vestibulum vaginae (1 out of 31; 3.2%), and testicular degeneration (4 out of 14; 28.6%) assessed by ultrasonographic investigations. The paucity of clinical signs associated with the high seroprevalence in the farm led to hypothesize that bovine besnoitiosis is endemic in the area though further studies are needed. Local practitioners should be more aware of the disease to facilitate the early detection of cases, prevent the spread of infection, and avoid economic losses and animal health problem.

Serum amyloid A levels and alpha 2 and gamma globulins on serum protein electrophoresis in cats exposed to and infected with Leishmania infantum.

BACKGROUND: Dogs are the main reservoir hosts of Leishmania infantum; nevertheless, recent investigations indicate a likely role for cats in the epidemiology of Leishmania infection. Feline leishmaniosis (FeL) remains poorly characterised, partly due to the lack of suitable diagnostic tools. This study aimed to compare serum amyloid A (SAA) levels and serum protein electrophoresis (SPE) profiles (specifically, alpha 2 and gamma globulins) in cats naturally exposed to or infected by L. infantum from southern Italy versus those of healthy controls and versus cats with neoplastic or inflammatory conditions from non-endemic areas. METHODS: Serum or plasma samples from four cohorts of cats were analysed for SAA levels and by SPE: (i) G1: healthy controls from Leishmania-non-endemic regions of Switzerland; (ii) G2: cats pre-diagnosed with neoplastic or inflammatory conditions available from the University of Cambridge sample archive; (iii) G3: L. infantum-seropositive, quantitative (q)PCR-negative cats from southern Italy; (iv) G4: L. infantum-seropositive and qPCR-positive cats from southern Italy. SAA data were assessed for normality and homoscedasticity using the Shapiro-Wilk and Levene's tests, respectively; the Kruskall-Wallis test, followed by Dunn's test with Bonferroni correction were subsequently used to compare SAA serum levels between groups. A weighted generalised linear model with a binomial distribution was used to assess statistically significant differences in the numbers of animals displaying elevated gamma globulins and increased alpha 2 globulins between groups. RESULTS: Overall, 68 samples were analysed (G1: n = 16, G2: n = 20, G3: n = 20, G4: n = 12). Cats suffering from neoplastic and inflammatory conditions (G2 ) showed significantly higher SAA levels than healthy controls (G1) (median values [interquartile range]: G1: 0.00 [0.00-0.00] mg/l versus G2: 0.85 [0.00-49.55] mg/l). G2, G3 and G4 cats showed higher percentages of individuals with increased alpha 2 globulins (percentages ± standard error: G1 = 20.0% ± 10.3, G2 = 80.0% ± 8.9, G3 = 70.0% ± 10.2, G4 = 75.0% ± 12.5) and gamma globulins (G1 = 0.0% ± 0, G2 = 65.0% ± 10.7, G3 = 50.0% ± 11.2, G4 = 58.3% ± 14.2) than healthy control cats (G1). For all three markers, no significant difference between cats within G2, G3 and G4 was recorded. CONCLUSIONS: This study indicates that the proportions of animals with elevated levels of alpha 2 and gamma globulins are significantly higher in cats exposed to and infected with L. infantum. Levels of SAA and alpha 2 and gamma globulins may not be used to differentiate between L. infantum infection or exposure, and neoplastic and/or inflammatory conditions.

A nationwide survey of Leishmania infantum infection in cats and associated risk factors in Italy.

Though scantly investigated, Leishmania infantum infection and clinical cases of leishmaniasis in cats have been recently reported in several countries of the Mediterranean basin, with large variability in prevalence data. A major limitation in the comparability of the data available is attributed to the differences in diagnostic techniques employed and cat populations sampled. The aim of this study was to assess the prevalence of L. infantum infection in owned cats across Italy by serological and molecular tests and the identification of potential risk factors. Blood samples from 2,659 cats from northern (n = 1,543), central (n = 471) and southern (n = 645) Italy were tested for antibodies against L. infantum, by an immunofluorescence antibody test and for the parasites' DNA, by real-time PCR. Samples were additionally screened for feline leukemia virus (FeLV) and feline immunodeficiency virus (FIV) proviral DNAs. An overall cumulative L. infantum prevalence of 3.9% was recorded by serology (3.3%) and/or qPCR (0.8%), with a higher rate (10.5%) in southern Italy. The risk of L. infantum infection in cats was significantly associated to the geographical areas (South vs North and Centre; p<0.0001), age class (from 19 months to 6 years old vs ≤18 months old, p = 0.0003), neutering status (not neutered vs neutered, p = 0.0028) and FIV infection (p = 0.0051).Though the role of cats in the epidemiology of L. infantum is still debated, our findings indicate that cats are exposed to and/or infected by this protozoan, mainly in endemic regions of Italy. Hence, a standardization of procedures for a prompt diagnosis of L. infantum infection in cats and for screening cat population is crucial for a better understanding of the epidemiology of feline leishmaniasis, and of the potential role of cats in the transmission cycle of zoonotic visceral leishmaniasis.

Serological survey and risk factors of Aelurostrongylus abstrusus infection among owned cats in Italy.

Feline lungworms affect the respiratory tract of domestic cats causing respiratory conditions of various degrees. In this study, we investigated the exposure of cats to feline lungworm infections by detecting antibodies in a large population of animals from several regions of Italy. Sera of 1087 domestic cats living in regions of the north (n = 700), the centre (n = 227) and the south (n = 160) of Italy were examined by a newly developed indirect ELISA conceived for detection of antibodies against the most frequently occurring feline lungworm Aelurostrongylus abstrusus. Individual cat data (i.e., age, sex, neutering status and provenience) were analysed as potential risk factors for exposure to lungworm infections. Samples were additionally screened for feline leukaemia virus (FeLV) and feline immunodeficiency virus (FIV) proviral DNAs. Overall, 9% (98/1087; 95% confidence interval (CI) 7.4-10.9%) of the animals tested seropositive to lungworm antibodies. Positive cats were identified in the north (7.1%; CI 5.5-9.3%), in the centre (5.3%; CI 3.0-9.0%) and in the South (22.5%; CI 16.7-29.6%), with more seropositive animals in the latter area (p < 0.05). The risk of lungworm infection in cats was significantly associated with age less than 6 months (i.e. 24.4%, p < 0.05) and FIV infection (p < 0.05). This large-scale serological survey confirms the exposure of cats to lungworm infections in Italy and that serological tests can be used to assess the distribution of lungworm infections in large populations of animals.

Treatment and long-term follow-up of a cat with leishmaniosis.

BACKGROUND: Leishmania infection in cats is being increasingly reported in endemic areas. Nevertheless, only a few clinical cases have been described in cats, and even fewer have provided information on the response to treatment and a proper follow-up. Here we report a case of feline leishmaniosis not associated with any other disease or co-infection and document its response to allopurinol treatment and long-term follow-up data. RESULTS: A 6-year-old domestic shorthair female cat was referred for nodular blepharitis, mucocutaneous ulcerative lesions of the mouth and lymph node enlargement. The cat was moderately anaemic, hyperglobulinaemic and tested negative for feline leukaemia virus and feline immunodeficiency virus. Fine needle aspirates of nodules and mucocutaneous lesions showed the presence of numerous amastigote forms of Leishmania. Leishmania infection was further confirmed by serology (IFAT test, 1:640) and real-time PCR (RT-PCR) on blood and conjunctival swabs. The cat was treated with allopurinol (20 mg/kg SID), which was clinically effective, although the cat remained Leishmania-positive in serology and RT-PCR on blood and conjunctival swabs. Allopurinol treatment was interrupted after seven months because of the healing of all lesions and lack of compliance by the owner. After two years, the cat relapsed displaying almost the same clinical signs and clinicopathological alterations. On this occasion, the parasite was isolated by culture and identified as belonging to L. infantum. Allopurinol treatment was started again but was interrupted several times because of the itching side effect observed. The cat worsened progressively and died two months after the relapse without any chance to shift the treatment to another molecule (e.g. meglumineantimoniate or miltefosine). CONCLUSIONS: Out of all documented cases of feline leishmanosis, the present case has the longest follow-up period and it is one of the few in which the parasite was isolated and identified. It further confirms the potential progression of Leishmania infection to disease in cats even in the absence of comorbidities. Veterinarians practicing in endemic areas should be aware of this susceptibility, properly include feline leishmaniosis in the differential diagnosis and propose preventative measures to those cats at risk.

Larval survival of Aelurostrongylus abstrusus lungworm in cat litters.

OBJECTIVES: The dehydrating power of cat litters may reduce the vitality of first-stage larvae (L1s) of feline lungworms, limiting copromicroscopical diagnosis. This study assessed the effect of the most commonly used cat litters on Aelurostrongylus abstrusus L1 survival. METHODS: Four types of cat litters were used: clumpling clay (group A); non-clumpling clay (group B); silica crystals (group C); and biodegradable (group D). A control group without litter (group E) was also included. On study day 0 (T0), L1s were obtained by the Baermann-Wetzel technique from the faeces of a naturally infected cat and ~100 larvae were injected in each of the 20 lungworm larvae-free faecal samples (~2 g each). Thereafter, four faecal samples per group were transferred into plastic cups containing the four different types of cat litters, or into empty cups (group E). The survival of L1s was assessed in each group after 3 (T3), 6 (T6), 12 (T12) and 24 (T24) h, using the Baermann-Wetzel technique. RESULTS: A decreasing trend of L1 survival was observed in all groups, with highest significant values at T0 compared with T3, T6, T12 and T24 (P <0.001). However, at T24, a significantly higher number (P <0.05) of L1s was extracted from faeces of the control group compared with the four groups with cat litters. CONCLUSIONS AND RELEVANCE: This study demonstrates how the survival of A abstrusus L1s, and therefore diagnosis, may be negatively influenced by the litter. The effect is time dependent, with a reduction in the number of vital larvae according to the type of litter, over time. False-negative results may be obtained, especially in cases of low parasitic load or when the sample is collected many hours after the emission.

Efficacy against nematode infections and safety of afoxolaner plus milbemycin oxime chewable tablets in domestic dogs under field conditions in Europe.

Afoxolaner (AFX) plus milbemycin oxime (MO) combination chewable tablets (NexGard Spectra®, Merial) were evaluated for safety and efficacy against naturally acquired nematode infections in domestic dogs in a multi-centre, positive control, blinded field study using a randomized block design based on the order of presentation for allocation. In total, 408 dogs confirmed positive for naturally acquired infections of intestinal nematodes by pre-treatment faecal examination were studied in ten countries in Europe (Albania, Austria, Bulgaria, France, Germany, Hungary, Italy, Lithuania, Romania and Slovakia). Pre-treatment faecal examination revealed Toxocara, Toxascaris, hookworm, Trichuris and/or Capillaria nematode infections in 134, 30, 223, 155 and 14 dogs, respectively. Dogs were allocated to one of two treatment groups in a ratio of 1, AFX + MO chewables (≥2.5 mg AFX + ≥0.5 mg MO per kg body weight, according to dose bands; 207 dogs), and 1, MO plus praziquantel (PRZ) chewables (Milbemax®, Novartis; ≥0.5 mg MO + ≥5 mg PRZ per kg body weight, according to the manufacturer's instructions; 201 dogs) and treated once. For evaluation of efficacy based on reduction of faecal nematode egg counts, two faecal samples, one collected prior to treatment and one collected 9 to 21 days after treatment, were examined using modified McMaster techniques. For evaluation of systemic safety, dogs were examined by a veterinarian before treatment administration and at study end, and dog owners observed the health status of their dogs until the end of the study and reported any abnormal observation. For dogs treated with AFX + MO chewables, the efficacy was 99.7, 99.7, 97.2, 99.7 and 99.7 % for Toxocara, Toxascaris, hookworm, Trichuris and Capillaria, respectively; and the efficacy was 99.5, 99.4, 94.3, 99.9 and 98.0 %, respectively, for the MO + PRZ-treated dogs (p ≤ 0.002 for all nematodes and both treatments). For Toxocara, hookworm and Trichuris, non-inferiority analysis demonstrated that the efficacy of AFX + MO chewable tablets was equal to or better than that of MO + PRZ. In spite that both treatments were ≥98 % efficacious against Toxascaris and Capillaria, a hypothesis of non-inferiority for both genera could not be established due to the low number of dogs infected with these parasites. No treatment-related adverse experiences were observed throughout the study. For both treatments, all dogs were given a systemic safety score of 'excellent' apart from one dog in each treatment group which received a score of 'acceptable'. AFX + MO combination chewables were shown to be safe and demonstrated a high level of efficacy when administered once to dogs infected with a broad range of parasitic nematodes under field conditions.

Efficacy of Broadline® spot-on against Aelurostrongylus abstrusus and Troglostrongylus brevior lungworms in naturally infected cats from Italy.

The increasing reports of Aelurostrongylus abstrusus infection and the new information on Troglostrongylus brevior have spurred the interest of the scientific community towards the research of pharmaceutical compounds effective against both pathogens. A novel topical combination of fipronil, (S)-methoprene, eprinomectin and praziquantel (Broadline®, Merial) has been released for the treatment of a variety of feline parasitic infections. The present study reports the efficacy of this spot-on in treating cats naturally infected by feline lungworms. Client owned cats (n=191) were enrolled from three geographical areas of Italy and faecal samples were examined by floatation and Baermann techniques. Twenty-three individuals were positive for L1 of A. abstrusus (n=18) or T. brevior (n=3) or for both species (n=2) and they were topically treated with Broadline®. Seventeen of them were also concomitantly infected by other parasites. Four weeks after treatment, faecal samples were collected and examined to assess the efficacy of a single administration of the product. Based on lungworm larvae counts, the efficacy of the treatment was 90.5% or 100% for A. abstrusus or T. brevior, respectively. Cats released significantly lower amounts of lungworm larvae after treatment compared to pre-treatment (p<0.0001). All but three cats were negative for other nematodes after treatment and all cats recovered from respiratory signs. Results of this study indicate that a single administration of the topical combination fipronil, (S)-methoprene, eprinomectin and praziquantel is effective and safe for the treatment of A. abstrusus and/or T. brevior infections in cats living under field conditions.

Feline lungworm Oslerus rostratus (Strongylida: Filaridae) in Italy: first case report and histopathological findings.

Oslerus rostratus syn. Anafilaroides rostratus (Strongylida: Filaroididae) is a metastrongyloid transmitted by snails, which localizes in peri-bronchial tissues and in the lung parenchyma of wild as well as domestic cats. In Europe, this nematode has been reported only on two occasions, being diagnosed in cats from Majorca Island and in northern Spain. Here, we describe a case of O. rostratus infection in a necropsied 4-year-old cat in Sicily (southern Italy). At the inspection of lungs, slender and greyish nematodes (four females and two males) were found embedded in the peri-bronchial tissues and in the bronchial walls. Parasites were morphological and molecularly identified as O. rostratus, with their 18S sequences being identical among them and showing a high homology (99%) with those available in public databases. At the histology, nematodes were encapsulated in a pseudo-cystic formation surrounded by an interstitial inflammatory process and fibrous tissue. Lung lesions were mainly represented by peri-luminal fibrosis, hyperplasia and hypertrophy of the bronchial mucosa and glands, respectively. This first record of O. rostratus infection from Italy indicates that this parasite should be included in the differential diagnosis of feline of lungworm infection.

Troglostrongylus brevior and Troglostrongylus subcrenatus (Strongylida: Crenosomatidae) as agents of broncho-pulmonary infestation in domestic cats.

BACKGROUND: Aelurostrongylus abstrusus is currently regarded as the main metastrongyloid infesting domestic cats, whereas the reports of Troglostrongylus spp. in domestic and wild felids largely remain anecdotic. This paper reports on pulmonary infestation caused by Troglostrongylus brevior and Troglostrongylus subcrenatus in two kittens and describes, for the first time, associated clinical presentations and pathological features. Morphometrical, molecular and phylogenetic analyses have also been conducted to differentiate here the examined Troglostrongylus species from A. abstrusus, towards a clearer delineation of metastrongyloids affecting cats. METHODS: Two kittens were referred for respiratory distress and hospitalized with a diagnosis of severe aelurostrongylosis, based on the presence of metastrongyloid larvae in the faeces. Despite prompt treatment, kittens died within 48 hours. Both kittens were submitted to necropsy to determine the cause of death. RESULTS: At necropsy, nematode specimens were found in the trachea, bronchi and bronchioles and were associated with respiratory signs (i.e., dyspnoea, polypnea, severe coughing and nasal discharge). Morphology and measurements of adult parasites found allowed the unequivocal identification of T. brevior and T. subcrenatus, even if first stage larvae were rather similar to those of A. abstrusus. Briefly, T. brevior and T. subcrenatus larvae were shorter in length and lacking the typical knob-like terminal end of A. abstrusus. Molecular and phylogenetic analyses corroborated morphological identification and provided data on mitochondrial and ribosomal DNA genes of T. brevior. CONCLUSIONS: Data presented here indicate that T. brevior and T. subcrenatus may cause major respiratory distress in domestic cats. Consequently, these two species should be included, along with A. abstrusus, in the differential diagnosis of cat bronchopulmonary affections and treatment protocols need to be evaluated. Through research on the biology, epidemiology and control of Troglostrongylus spp. infestations in domestic cats are advisable to implement current knowledge on these neglected metastrongyloids.