Plasma markers of inflammation and hemostatic and endothelial activity in naturally overweight and obese dogs.

BACKGROUND: Obesity is one of the most prevalent health problems in the canine population. While haemostatic parameters and markers of endothelial function have been evaluated in various disease conditions in dogs, there are no studies of these markers in canine obesity. This study was designed to evaluate the effect of naturally gained weight excess and obesity on inflammatory, hemostatic and endothelial biomarkers in dogs. A total of 37 overweight and obese dogs were compared with 28 normal weight dogs. RESULTS: Overweight and obese dogs had significantly elevated concentrations of serum interleukin-6 (IL-6) and C-reactive protein (hsCRP). Number of platelets, activity of factor X and factor VII were significantly higher, while activated partial thromboplastine time (aPTT) and soluble plasminogen activator receptor (suPAR) were significantly decreased. Statistical analysis of high mobility group box - 1 protein (HMGB-1), soluble intercellular adhesive molecule -1 (sICAM-1) and plasminogen activator inhibitor type 1 (PAI-1) concentrations did not show significant differences between the total overweight and obese group and the normal weight group of dogs. CONCLUSIONS: Analytical changes in the dogs in our study reflects that weight excess in dogs can be associated with a chronic low degree of inflammation and a hypercoagulable state, where primary and secondary hemostasis are both affected. However obesity is not associated with impairment of endothelial function in dogs.

Liver enzymes and blood metabolites in a population of free-ranging red deer (Cervus elaphus) naturally infected with Fascioloides magna.

We investigated the effects of Fascioloides magna infection on the serum biochemistry values of the naturally infected red deer population in eastern Croatia. The investigation was performed on 47 red deer with F. magna infection confirmed patho-anatomically in 27 animals (57.4%). Fibrous capsules and migratory lesions were found in 14 deer while only fibrous capsules without migratory lesions were found in 13 deer. In 13 deer both immature and mature flukes were found, in 5 deer only immature flukes were found and in 9 deer only mature flukes were found. Fascioloides magna infected deer with fibrous capsules and migratory lesions had significantly higher values for lactate dehydrogenase (LDH), glutamate dehydrogenase (GLDH) and globulin, and lower values for albumin/globulin ratio and glucose compared to uninfected deer. Fascioloides magna infected deer with fibrous capsules without the presence of migratory lesions had higher values for alanine aminotransferase (ALT) and globulin, and lower values for albumin/globulin ratio and glucose, than the uninfected deer. The number of immature flukes was positively correlated with values of aspartate aminotransferase (AST), γ-glutamyltransferase (GGT), LDH, GLDH, urea and triglycerides. The number of migratory lesions was positively correlated with GGT, GLDH, globulin and urea values. The creatinine value was positively correlated with the number of mature flukes. The trial showed that F. magna infection causes significant changes in serum biochemistry. Moreover, these changes do not completely resemble changes following F. hepatica infection. Further investigation of changes in liver enzymes and other serum metabolites in controlled, experimentally induced fascioloidosis in red deer is needed to better understand the pathogenesis of F. magna.

Validation of a Western Blot for the detection of anti-Trichinella spp. antibodies in domestic pigs.

Trichinellosis is a zoonotic disease in humans caused by Trichinella spp. According to international regulations and guidelines, serological surveillance can be used to demonstrate the absence of Trichinella spp. in a defined domestic pig population. Most enzyme-linked immunosorbent assay (ELISA) tests presently available do not yield 100% specificity, and therefore, a complementary test is needed to confirm the diagnosis of any initial ELISA seropositivity. The goal of the present study was to evaluate the sensitivity and specificity of a Western Blot assay based on somatic Trichinella spiralis muscle stage (L1) antigen using Bayesian modeling techniques. A total of 295 meat juice and serum samples from pigs negative for Trichinella larvae by artificial digestion, including 74 potentially cross-reactive sera of pigs with other nematode infections, and 93 meat juice samples from pigs infected with Trichinella larvae were included in the study. The diagnostic sensitivity and specificity of the Western Blot were ranged from 95.8% to 96.0% and from 99.5% to 99.6%, respectively. A sensitivity analysis showed that the model outcomes were hardly influenced by changes in the prior distributions, providing a high confidence in the outcomes of the models. This validation study demonstrated that the Western Blot is a suitable method to confirm samples that reacted positively in an initial ELISA.

Multilocus genotyping of Giardia duodenalis reveals striking differences between assemblages A and B.

Giardia duodenalis is a widespread parasite of mammalian species, including humans. Due to its invariant morphology, investigations of aspects such as host specificity and transmission patterns require the direct genetic characterisation of parasites from faecal samples. We performed a sequence analysis of four genes (ssrRNA, ß-giardin, glutamate dehydrogenase and triose phosphate isomerase) of 61 human isolates and 29 animal isolates. The results showed that multilocus genotypes (MLGs) can be readily defined for G. duodenalis isolates of assemblage A but not for assemblage B. Indeed, for assemblage A isolates, there was no evidence of intra-isolate sequence heterogeneity, and congruent genotyping results were obtained at the four genetic loci investigated. Sequence comparison and phylogenetic analysis showed that human-derived and animal-derived MLGs are different, and further indicated the presence of a new sub-assemblage (referred to as "AIII"), which was found exclusively in wild hoofed animals. On the other hand, there were variable levels of intra-isolate sequence heterogeneity (i.e., the presence of two overlapping nucleotide peaks at specific positions in the chromatograms, or "heterogeneous templates") in assemblage B isolates from humans and animals, and this prevented the unambiguous identification of MLGs. Furthermore, in five human isolates and one non-human primate isolate, the assignment to assemblage B was problematic, given that one of the four markers supported an assignment to assemblage A. These findings raise concerns about the interpretation of genotyping data based on single markers, and indicate the need to understand the mechanisms that are responsible for the differences between G. duodenalis assemblages A and B.

A case of visceral leishmaniosis in a gray wolf (Canis lupus) from Croatia.

The southern habitats of Croatia's gray wolf (Canis lupus) population are found in central and southern parts of Dalmatia. This region is recognized as an endemic region for canine visceral leishmaniosis, caused by Leishmania infantum. In November 2003, a 4-yr-old male gray wolf was found dead in the northwestern border of this endemic region. Pathologic and parasitologic analysis, confirmed by polymerase chain reaction, indicated that lesions associated with infection by Leishmania infantum are, in this case, typical for visceral leshmaniosis commonly described in dogs. Review of the literature suggests that this is the first reported case of gray wolf death due to lesions caused by L. infantum.

First finding of libyostrongylosis in farm-reared ostriches (Struthio camelus) in Croatia: unusual histopathological finding in the brain of two ostriches, naturally infected with Libyostrongylus douglasi.

In the present work, the very first finding of Libyostrongylus douglasi in farm-reared ostriches in Croatia, was described, not only as the main cause of the death, but also as the cause of persistent infection. The results of histopathological examination of almost all parenchymatous organs in two routinely necropsied ostriches were presented, including atypical histopathological finding in the brain and the result of the L. douglasi scanning electron microscopical (SEM) examination. In order to determine the parasite species to which the larval form found in the brain belonged, polymerase chain reaction (PCR) was performed. Total DNA was isolated from fresh L. douglasi, and from archival formalin-fixed and paraffin-embedded brain section. Additionally, the smears of the proventricular mucosal lining were cytologically examined. Virological examination for newcastle disease virus (NDV) was also performed. As there was very limited information concerned ostrich's health status in Croatian farms, a preliminary evaluation of the parasite infestation level in the Croatian ostrich population over the period 2001-2002 was also done, and an attempt at characterising individual parasite species, was made.

Comparison of trichinelloscopy with a digestion method for the detection of Trichinella larvae in muscle tissue from naturally infected pigs with low level infections.

The identification of Trichinella infection in pigs in Croatia has traditionally been done by inspection of individual carcasses. In response to outbreaks of human trichinellosis in the last decade, the Croatian Ministry of Agriculture and Forestry instituted compulsory trichinelloscopic examination of tissue from both commercially and privately slaughtered swine. The purpose of this study was to compare trichinelloscopy and artificial digestion for use in samples containing low numbers of larvae. Each assay was used to test 1,769 field positive samples, 290 of which contained 6 or less larvae per gram of muscle tissue. The sensitivity and specificity of trichinelloscopy with 6 or less l pg was 43.4 and 88%, respectively. kappa-Value as a measure of agreement between trichinelloscopy and artificial digestion was 0.27%. It is noteworthy that a considerable number of the 103 (52%) negative animals on trichinelloscopy contained>or=6l pg which is enough to cause clinical trichinellosis. These findings support other studies that indicate trichinelloscopy is not a method of choice and that it is necessary to implement more sensitive procedures such as artificial digestion.

Evaluation of ELISA for detection of Trichinella antibodies in muscle juice samples of naturally infected pigs.

The performance characteristics of an ELISA test for trichinellosis in pigs applied to muscle juice was assessed using 314 samples collected from pigs located in endemic areas of Croatia. Peptic digestion was used as the reference method. The diagnostic accuracy of the two compared dilutions (1:10 and 1:100) was considered to be high because the area under the curve (AUC) index was 0.922 and 0.920 for each dilution, respectively. In this study the two graph-receiver operating characteristic (TG-ROC) analysis was used as a tool for selecting cut-off points. Sensitivity, specificity, likelihood ratios, efficiency and Youden's index were used as indices of test accuracy. The cut-off values that minimize overall misclassification cost under an assumption of 3% prevalence were calculated. Our results indicate that the ELISA applied to muscle juice is a highly accurate test and can be adapted to process a large number of samples.

A seroepidemiologic survey of canine visceral leishmaniosis among apparently healthy dogs in Croatia.

Cross-sectional investigation was done on seroprevalence of Leishmania sp. infection among apparently healthy dogs in an area where canine leishmaniosis is endemic. Survey included 68 dogs living in the coastal city of Split, and 238 dogs living in 12 villages scattered in the hinterland. Each dog was clinically examined for the presence of some discrete signs compatible with leishmaniosis and by dot-ELISA modification determined the presence of anti-Leishmania antibodies. The titre 1:600 and higher was regarded as positive in the study. The seroprevalence ranged from 0 to 42.85%, depending on the location. 54.34% of the seropositive dogs had moderately enlarged lymph nodes and/or some discrete changes on the skin. In our parasitological study, Leishmania sp. was isolated from several seropositive animals that had some clinical signs and from a few which did not have any. Data analysis revealed that serological positivity to Leishmania sp. was not associated with a dog's outdoor lifestyle and utility, but was associated with the gender and age.

Influence of methods for Trichinella detection in pigs from endemic and non-endemic European region.

A total of 1401 German and 226 Croatian pigs raised either indoors or outdoors were tested for Trichinella infection by direct and indirect detection methods. A 10 g sample of diaphragm were examined for muscle larvae by the artificial digestion method; the species was determined by multiplex polymerase chain reaction (PCR). For detection of anti-Trichinella IgG, serum samples diluted 1:100, and meat juice samples diluted 1:10, were tested by enzyme-linked immunosorbent assay. All German pigs and those Croatian pigs raised indoors proved to be Trichinella-negative by all methods. Muscle larvae were detected in a total of eleven of the Croatian pigs, which were raised on small outdoor farms. For eight isolates, PCR results demonstrated that recovered larvae were Trichinella spiralis. Anti-Trichinella-IgG was detected in serum and meat juice of digestion positive animals when the worm burdens exceeded 0.38 larvae per gram of muscle. Positive results in Croatian pigs indicate a higher risk of infection for outdoor farming in areas where Trichinella is endemic. Results of direct and indirect detection were compared and are discussed with special regard to specificity and sensitivity of methods.

Stress-induced Cushing's syndrome in fur-chewing chinchillas.

One of the most serious problems in the chinchilla industry is 'fur-chewing', when the chinchilla bites off areas of its own or some other animal's fur. The condition generally develops in both genders at the age of 6-8 months. In chinchilla farms in Croatia an incidence of 15-20% has been observed. A pathomorphological, microbiological and parasitological investigation was conducted on eleven 6- to 11-month-old chinchillas of both sexes with clinical symptoms of 'fur-chewing' and three chinchillas without such signs. Histopathology of the adrenal glands and of the chewed skin revealed changes typical of Cushing's syndrome in 'fur-chewed' chinchillas, such as hyperkeratinisation of the epidermis, epidermal atrophy, pronounced follicular and sebaceous gland atrophy, hyperkeratinisation of the follicles with comedo formations and the presence of calcium salts in subcutis.

The efficacy of flubendazole against Trichinella spiralis in swine.

A trial was undertaken to assess the efficacy of flubendazole against experimentally induced trichinellosis in pigs when given mixed with food at different dose rates. At the start of the experiment, 48 pigs were infected with approximately 35,000 larvae of Trichinella spiralis, afterwards the animals were distributed in six groups. The first three groups were used to test the activity of the drug against adult stages. Other three groups served to define the efficacy against muscle larvae. The dose rate of flubendazole varied according to the purpose of the experiment (from 8 mg/kg up to 62 mg/kg). At the end of the trial, the pigs were necropsied and the number of adults and larvae in predilection muscles were determined. At a dose rate of 8 mg/kg flubendazole supplied with food during eight days was 100% effective against adults. At a dose rate of 31 mg/kg flubendazole administered for 14 days was 72.35% effective. Treatment with higher dosages of flubendazole (62 mg/kg) resulted in increased efficacy (87.77%) against the parasites. The biological assay performed with larvae from the muscle samples originating from the pigs treated with 62 mg/kg showed that at least half of the larvae were not infective.

Epidemiology of swine trichinellosis in the Republic of Croatia.

In order to study the epidemiology of trichinellosis with special emphasis on swine a detailed study was performed. The goals of the study were to define the prevalence of trichinellosis in swine and rats and to determine the etiological agents of the disease with special emphasis on sylvatic species, Trichinella britovi. Considering the obtained data it can be concluded that the risk of Trichinella infection for swine in endemic areas in Croatia is rather high today. On the contrary, a sharp decline in human cases is registered what is certainly a result of obligatory trichinelloscopy even for private purposes. The parasite was found to be prevalent in rats too. Studies on genotypes suggest that Trichinella spiralis is the most prevalent etiological agent of domestic trichinellosis in Croatia, even so the sylvatic species Trichinella britovi can be also present in the domestic habitat suggesting a link between the sylvatic and domestic cycle.

Analysis of intestinal intraepithelial lymphocyte populations in experimental Trichinella spiralis infection of mice.

The potential role of intestinal intraepithelial lymphocytes (i-IELs) in the generation of host protective immunity after helminth infection was investigated using the Trichinella spiralis (Owen, 1835)/mouse model. In this study we found a significant rise of TCRgamma(delta)+ i-IELs (P < 0.001) concurrent with the jejunal goblet cells (GC) hyperplasia in T. spiralis-infected C57BL mice on day 4 p.i. However, no direct relationship between the kinetics of the increase in TCRgamma(delta)+ i-IELs and T. spiralis expulsion was observed in infected mice. Taken together, these results implicate that gamma(delta) i-IELs probably perform a unique functions related to the regulation of the GC proliferation accompanying T. spiralis gut infection. As is known, these TCRgamma(delta)+ i-IELs may release mediators or growth factors that in turn influence GC differentiation. With the use of dexamethason (DEX), a potent anti-inflammatory agent which also induces apoptotic cell death in i-IELs, we have confirmed that the expulsion of T. spiralis from the mouse gut is accompanied by an inflammatory response. Indeed, the GC are clearly involved in these phenomena, apparently under the regulation by TCRgamma(delta)+ i-IEL-mediated responses, since DEX abrogated GC proliferation in T. spiralis-infected C57BL mice and subsequently augmented adult worm burden. Our data also show that the rejection of adult worms starts concurrently with a significant increase in TCRalpha(beta)+ and CD8+ i-IELs (P < 0.05 and P < or = 0.01, respectively), namely by day 7 p.i. At the same time, CD4+ cells significantly decreased (P < 0.05) in the intestinal epithelium of T. spiralis-infected, vs uninfected mice. These results may indicate that the TCRalpha(beta)+ and CD8+ i-IELs act as effectors of anti-T spiralis defence reactions. The implications of these findings for the potential role of intestinal intraepithelial CD8+ and TCRalpha(beta)+ cells in the pathogenesis of the intestinal lesions during T. spiralis gut infection are discussed.

gamma delta TCR+ intestinal intraepithelial lymphocytes (i-IEL) in reaction against intestinal nematode Trichinella spiralis.

To assess the gamma delta TCR T cells in the control of the timing of the mucosal response to enteric parasitic infections, we used C57BL mice, orally infected with 200 viable T. spiralis larvae. The small intestine, spleens and Peyer's patches (PP) were excised on 1, 4, 7, 14, 21 and 29 postinfection days (p.i.) for immunophenotyping and histological studies. Uninfected mice served as control. Characterization of isolated lymphocytes of C57BL control mice, confirmed that T cell immunophenotype differs in spleen, PP and i-IEL. Practically all i-IEL were CD3+ cells (83%). In addition, most of the i-IEL expressed Ly-2 (65%). Among the i-IEL, the level of gamma delta TCR+ cells was significantly higher (29%) than that found in spleen (3%) and PP (3%). The expression was high on CD3+ and Ly-2+ (26 and 21%, respectively) and low on L3T4+ i-IEL (< 1%). During T. spiralis infection alpha beta TCR+ CD3+, gamma delta TCR+ CD3+ and gamma delta TCR+ Ly-2+ i-IEL increased on day 4 and 7. However, infected mice displayed a reduction in i-IEL number from 14 to 29 p.i. day. At the same time the proportion of gamma delta TCR on spleen Ly-2+ and on PP CD3+ and Ly-2+ cells increased on 14 and 21 p.i. day. Adult worms were expelled from the gut by day 14. Thus, the kinetics of gamma delta TCR+ i-IEL, but not spleen and PP gamma delta TCR, corresponded to the kinetics of worm expulsion in C57BL mice. Most murine i-IEL of the gamma delta T cell lineage tend to be cytolytic when activated. We speculated that gamma delta T cells of i-IEL during the early stages of infection recognize and eliminate damaged epithelial cells generated by parasite antigens, simultaneously accelerating the worm expulsion.

Immunosuppression of in vivo and in vitro lymphocyte responses in swine induced by Trichinella spiralis or excretory-secretory antigens of the parasite.

The in vivo and in vitro effects of Trichinella spiralis excretory-secretory (ES) antigens on porcine peripheral blood lymphocyte (PBL) responses induced with mitogens (phytohemagglutinin, PHA; concanavalin A, Con A; pokeweed mitogen, PWM) or unrelated antigen (Protein A) were studied to determine whether ES antigens depress lymphocyte responses in experimental swine trichinosis, and/or if this response was manifested after lymphocytes from infected pigs had been pretreated with ES antigens. Additionally, the range of inhibition of lymphocyte responses was tested in parasite-free pigs using different doses of ES antigens and compared with the responsiveness of control cultures from the same animals. The responses of lymphocytes from pigs inoculated with 4 x 10(3) muscle larvae (ML) were strongly depressed (P < 0.05) at post-inoculation days (PID) 7 (after stimulation with PHA), 14, 35 (Con A or PWM), and 49 (PWM). At PID 56 and 63 the lymphocytes from T. spiralis-infected pigs responded better (P < 0.05) to all three mitogens than those from non-infected controls. After 7 weeks post-inoculation, PBL which were pretreated with 10 or 250 micrograms ml-1 of ES antigens showed significantly weaker (P < 0.05, P < 0.001) responses to PWM or PHA, respectively, than those from non-infected animals. The responsiveness of lymphocytes from both groups of pigs to Protein A was not affected by the pretreatment with ES antigens in vitro. The responses of lymphocytes from the parasite-free pigs induced by PHA, PWM or Protein A were strongly depressed (P < 0.01) after in vitro pretreatment regardless of the dose of ES antigens (5, 10, 15, or 20 micrograms ml-1) applied.

Epidemiological survey of trichinellosis in Yugoslavia.

In a long term epidemiological survey that included different hosts for Trichinella spiralis among wild and domestic animals, the parasitological and serological screening revealed a high number of positive animals. The incidence of T. spiralis in grazing pigs was high, 21.76% (170 examined) but most of positive animals were lightly infected (0.016-0.02 l/g). While out of 2,394 pigs raised in small private farms, 1.67% were positive with high infection rates, none of the pigs raised on a modern breeding and fattening farm were seropositive. A repeated survey in grazing pigs performed five years later and in the same area, showed the prevalence of seropositive pigs to be still high (22% of 100 examined pigs). The same data were confirmed by detection of specific anti-Trichinella antibodies in Western blotting procedure. One of 2,031 serologically (IgG-ELISA) examined horses from Croatia reacted positively with very high OD values. Different characteristics of biological patterns (RCI, female fecundity, capsule formation, isoenzyme analysis) were found between two well distinguished groups of isolates. The first group consisted of well defined T1 isolates with patterns described by the Reference Centre for Trichinellosis, Rome, Italy. The data obtained by isoenzyme analysis of the representative sylvatic isolate were comparable to T3 characteristics of isolates from foxes in Italy.

Immunity in swine inoculated with larvae or extracts of a pig isolate and a sylvatic isolate of Trichinella spiralis.

Inoculation of swine with a sylvatic isolate of Trichinella spiralis, designated T s nativa, resulted in low numbers of muscle larvae, compared with muscle larvae accumulation in swine inoculated with a pig type of T s spiralis. Despite low infectivity of T s nativa for swine, primary inoculation resulted in high levels of immunity against challenge infection with T s spiralis. This immunity was expressed in accelerated expulsion of challenge adults from the intestine and reduced numbers of muscle larvae. Pigs inoculated with T s nativa developed cellular and humoral responses similar to those in pigs inoculated with T s spiralis. However, in immunoblots, sera from pigs inoculated with T s nativa recognized additional proteins in muscle larvae excretory-secretory (ES) products, compared with sera from pigs inoculated with T s spiralis. Active immunization of pigs with ES products from T s nativa resulted in numerically higher, but not significantly different levels of immunity, compared with pigs immunized with ES from T s spiralis. The highest levels of immunity were obtained in pigs immunized with a T s spiralis newborn larval extract. The combination of ES products and newborn larval extract did not result in additive levels of immunity. These results indicate that the major immune effector response to Trichinella sp in pigs is against the newborn larvae, regardless of the genetic type of Trichinella sp.

Characterization of a noncyst-forming isolate of Trichinella from a wild boar in Yugoslavia.

An isolate of Trichinella obtained from a wild boar in Yugoslavia did not form cysts in the musculature of its natural host. Subsequent inoculation into experimental hosts demonstrated that some larvae became encysted only after extended time periods, whereas others remained unencapsulated. Histological staining of larvae in the musculature demonstrated no deposition of collagen typically seen for Trichinella spiralis spiralis, Trichinella spiralis nativa, or Trichinella spiralis nelsoni. The Yugoslavian isolate, given the name of Zagreb isolate after the University where it was first studied, had low infectivity for pigs and mice. Isozyme analysis demonstrated greater homology with T. s. nelsoni than with other subspecies of Trichinella. Restriction fragment length polymorphisms and dot blot analyses further demonstrated the distinctive nature of this isolate. These results suggest that lack of cyst formation might be characteristic of isolates other than those designated Trichinella pseudospiralis and that this character might be important in the classification of Trichinella.

Evaluation of excretory-secretory antigens for the serodiagnosis of swine trichinellosis.

Groups of hog sera from endemic and non-endemic areas for swine trichinellosis in Yugoslavia were tested by ELISA using excretory-secretory (ES) antigens collected from T. spiralis muscle larvae maintained in vitro for 24, 48 or 72 h. The 24-h ES had the highest level of specificity for T. spiralis infection. Antigen preparations recovered after 48 or 72 h yielded an increasing rate of false-positive reactions. Additional antigens occurred in the 48- and 72-h ES preparations as determined by gel electrophoresis and monoclonal antibody binding. The occurrence of false-negative reactions was directly correlated with T. spiralis worm burdens. Hogs with muscle larvae densities greater than 10 larvae per gram were all positive by ELISA. Among 17 hogs with less than 10 larvae per gram, only one hog was negative by ELISA with 24-h ES antigen; the false-negative rate was higher with 48- and 72-h ES. These results show that ES antigen produced during the first 24 h of in vitro cultivation is highly specific for the immunodiagnosis of swine trichinellosis.