Screening of microfilaricidal effects of plant extracts against Dirofilaria immitis

Canine dirofilariasis is a common tropical parasitic disease of companion animals, caused by infestation of Dirofilaria immitis filarids within the pulmonary arteries and extending into the right heart. Increased reports of adverse reactions elicited by current microfilaricidal agents against D. immitis such as neurological disorders, circulatory collapse and potential resistance against these agents, warrant the search for new agents in forms of plant extracts. The use of plant extracts in therapeutic medicine is commonly met with scepticism by the veterinary community, thus the lack of focus on its medical potential. This study evaluated the presence of microfilaricidal activities of the aqueous extracts of Zingiber officinale, Andrographis paniculata and Tinospora crispa Miers on D. immitisin vitro at different concentrations; 10 mg/ml, 1 mg/ml, 100 μg/ml, 10 μg/ml and 1 μg/ml within 24 h, by evaluation of relative microfilarial motility as a measure of microfilaricidal activity. All extracts showed microfilaricidal activity with Z. officinale exhibiting the strongest activity overall, followed by A. paniculata and T. crispa Miers. It is speculated that the microfilaricidal mechanism exhibited by these extracts is via spastic paralysis based upon direct observation of the microfilarial motility.     

Quantitative real-time PCR and culture examination of Mycobacterium avium subsp. paratuberculosis at farm level

Mycobacterium avium subsp. paratuberculosis (MAP) causes Johne's disease in ruminants and may contribute to Crohn's disease in humans. The aim of this study was to determine the occurrence and quantity of MAP in cattle feces and milk in the Iranian context. In addition, we evaluated the effect of cattle age as well as farming system as risk factors contributing to MAP load. For this, a total sample of 373 consisting of 150 cattle feces (CF), 150 individual cow's milk (ICM), as well as 73 bulk-tank milk (BTM) was collected randomly and regardless of the cattle health status. The samples were assayed using F57 quantitative real-time PCR (qPCR) and culture method. According to the results of qPCR which was found ∼10 times more sensitive than culture assay, MAP was detected in 68.66% (103/150) of the CF, 12% (18/150) of the ICM and 52.05% (38/73) of the BTM samples. In contrast to the previous reports, the quantity of MAP in the BTM (2.03–5.97 log cfu/50 ml) was statistically (p < 0.01) higher than the ICM (0.90–1.97 log cfu/50 ml). Data suggested a direct relation (p < 0.01) between the cattle age and the quantity of MAP in the CF samples, while the relation was not statistically significant (p > 0.05) for the ICM. In addition, MAP load in the BTM samples obtained from traditional farms was significantly (p < 0.01) higher than that of the industrial ones, while the differences in CF and ICM was not significant (p > 0.05).     

Detection and molecular characterization of Theileria sp. in fallow deer (Dama dama) and ticks from an Italian natural preserve

The prevalence of piroplasms in a closed population of fallow deer (Dama dama L.) living in the Italian preserve of “Bosco della Mesola” - Ferrara (Mesola wood) was investigated. Blood samples and ticks were collected from 62 fallow deer. On microscopic observation, 28 (45.0%) blood samples were positive for piroplasms while PCR provided evidence for piroplasms infection in 47 (75.8%) fallow deer. The 67 ticks, collected from positive and negative animals, were identified as Ixodesricinus L., 1758 (89.6%) and Haemaphysalisconcinna Koch, 1844 (10.4%). At the PCR, four samples of I. ricinus were positive for piroplasms. The sequences of the 18S rRNA gene from both blood and ticks were identical and showed high identity (99.6%) with Theileria sp. 3185/02 (DQ866842) and Theileria capreoli (AY726011) from roe deer. Interestingly, the phylogenetical analyses evidenced differences between the Theileria strain from Mesola wood and the ones isolated in fallow deer from other Italian areas.     

Effectiveness of small interfering RNA (siRNA) against the Mcl-1 gene in a canine mammary gland tumor cell line

The effect of down-regulation of Mcl-1 expression by small interfering RNA (siRNA) against the canine Mcl-1 gene on apoptosis was investigated by transfecting CF33 (canine mammary gland tumor cell line) with siRNA using cationic liposomes. The siRNA against canine Mcl-1 increased the rate of apoptotic cells and decreased the numbers of viable cells. Further, sequence-specific down-regulation of Mcl-1 expression was measured by real time-PCR and Western blot analysis. The siRNA directed against the Mcl-1 gene reduced both the mRNA and protein expression in the CF33. Our study suggests the importance of Mcl-1 in canine mammary tumors for inducing apoptosis and reinforces using Mcl-1 as a putative therapeutic target in canine mammary gland tumor.     

Prevalence and antimicrobial susceptibility of Salmonella spp. in small Indian mongooses (Herpestes auropunctatus) in Grenada,West Indies

Intestinal samples from 156 small Indian mongooses (Herpestes auropunctatus) collected island-wide in Grenada from April 2011 to March 2013 were examined for the presence of Salmonella enterica spp. Nineteen (12%) mongooses were culture-positive for S. enterica spp. of which five serotypes were identified. Salmonella javiana and S. Montevideo were the most commonly isolated serotypes. The other serotypes isolated were S. Rubislaw, S. Panama and S. Arechavaleta. All isolates were susceptible to amoxicillin–clavulanic acid, ampicillin, cefotaxime, ceftazidime, ciprofloxacin, enrofloxacin, gentamicin, nalidixic acid, imipenem and trimethoprim–sulfamethoxazole. One isolate (S. Montevideo) showed resistance to tetracycline and intermediate resistance to streptomycin. The five isolated Salmonella serotypes are potential human pathogens suggesting that the mongoose may play a role in the epidemiology of human salmonellosis in Grenada.     

Association of CXCR1 polymorphisms with apoptosis,necrosis and concentration of milk neutrophils in early lactating dairy heifers

Associations between polymorphisms in the candidate gene CXCR1, encoding the chemokine (C-X-C motif) receptor 1, and udder health have been identified before. In the present study, associations between the CXCR1 genotype (whole coding region) and apoptosis, necrosis, and concentration of milk polymorphonuclear neutrophilic leukocyte (PMNL) of 292 quarters belonging to 73 early lactating dairy heifers were studied. In uninfected quarters, % milk PMNL apoptosis was higher in c.980GG heifers [least squares means (LSM) 27%] compared to c.980AG heifers (LSM 16%), whereas in infected quarters, % milk PMNL apoptosis was higher in c.642GG heifers (LSM 29%) compared to c.642AG heifers (LSM 18%). Differences in milk PMNL concentration between infected and uninfected quarters were smaller in c.980AG heifers than in c.980GG heifers. An association between the CXCR1 genotype and necrosis of milk PMNL could not be demonstrated. Results indicate that CXCR1 polymorphisms influence viability and concentration of milk PMNL and provide a foundation for future research.     

A zoospore inhibition technique to evaluate the activity of antifungal compounds against Batrachochytrium dendrobatidis and unsuccessful treatment of experimentally infected green tree frogs (Litoria caerulea) by fluconazole and benzalkonium chloride

Effective and safe treatments of chytridiomycosis in amphibians, caused by Batrachochytrium dendrobatidis, are needed to help prevent mortality in captive programs for threatened species, to reduce the risk of spread, and to better manage the disease in threatened populations. We describe a simple method to determine minimum inhibitory concentrations (MICs) of antifungal agents that involves adding zoospores to various drug concentrations in 96 well plates and microscopic observation after four days. We report results from testing 10 commercially available antifungal compounds: benzalkonium chloride (<0.78 μg/ml), povidone iodine (312.5 μg/ml), amphotericin B (3.125 μg/ml), fluconazole (<1.56 μg/ml), itraconazole (<1.56 μg/ml), enilconazole (<1.56 μg/ml), mercurochrome (6.25 μg/ml), sodium chloride (12.5 mg/ml), methylene blue (<1.56 μg/ml) and Virkon (3.125 μg/ml). For treatment trials of juvenile Litoria caerulea, baths of benzalkonium chloride at 1 mg/L and fluconazole at 25 mg/L were used on 18 experimentally infected frogs per treatment. Although these treatments resulted in longer survival times (mean 43.7 ± 11.3 days) than in the untreated controls (37.9 ± 9.3 days), the mortality rate was still 100%. Higher doses of fluconazole are suggested for further animal trials.     

The efficacy of eprinomectin extended-release injection against Hypoderma spp. (Diptera: Oestridae) in cattle

The efficacy of eprinomectin in an extended-release injection (ERI) formulation was determined in cattle harboring naturally acquired infestations of first- or second- and third-stage larvae of Hypoderma spp. in three studies conducted according to the same protocol in the USA (two studies) and Germany (one study). Thirty cattle sourced from herds with a history of Hypoderma infestation were included in each study. Cattle were formed into replicates of three animals each on the basis of pre-treatment anti-Hypoderma antibody titers. Within replicates each animal was randomly allocated to one of the following treatments: ERI vehicle (control) at 1 mL/50 kg bodyweight, administered once on Day 0; Eprinomectin 5% ERI at 1 mL/50 kg bodyweight (1.0 mg eprinomectin/kg), administered once on Day 0 (when larvae were expected to be first instars); or Eprinomectin 5% ERI at 1 mL/50 kg bodyweight (1.0 mg eprinomectin/kg), administered once when larvae were second or third instars (study dependent, Day 73, 119, or 140). Treatments were administered by subcutaneous injection in front of the shoulder. In all studies, emerging and/or expressed Hypoderma larvae were recovered, speciated, and counted and viability was determined. Eprinomectin LAI treatment was 100% (p < 0.05) efficacious against first- and second- or third-stage larvae of Hypoderma bovis (two studies) and Hypoderma lineatum (one study). All animals accepted the treatment well. No adverse reaction to treatments was observed in any animal in any study.     

Horizontal transmission dynamics of White spot syndrome virus by cohabitation trials in juvenile Penaeus monodon and P. vannamei

White spot syndrome virus (WSSV), a rod-shaped double-stranded DNA virus, is an infectious agent causing fatal disease in shrimp farming around the globe. Within shrimp populations WSSV is transmitted very fast, however, the modes and dynamics of transmission of this virus are not well understood. In the current study the dynamics of disease transmission of WSSV were investigated in small, closed populations of Penaeus monodon and Penaeus vannamei. Pair cohabitation experiments using PCR as a readout for virus infection were used to estimate transmission parameters for WSSV in these two species. The mortality rate of contact-infected shrimp in P. monodon was higher than the rate in P. vannamei. The transmission rate parameters for WSSV were not different between the two species. The relative contribution of direct and indirect transmission rates of WSSV differed between the two species. For P. vannamei the direct contact transmission rate of WSSV was significantly lower than the indirect environmental transmission rate, but for P. monodon, the opposite was found. The reproduction ratio R₀ for WSSV for these two species of shrimp was estimated to be above one: 2.07 (95%CI 1.53, 2.79) for P. monodon and 1.51 (95%CI 1.12, 2.03) for P. vannamei. The difference in R₀ between the two species is due to a lower host mortality and hence a longer infectious period of WSSV in P. monodon.     

Anthelmintic efficacy of neem (Azadirachta indica A. Juss) and the homeopathic product Fator Vermes in Morada Nova sheep

Gastrointestinal nematodes are becoming increasingly resistant to the commercial products used to control them. The cost of routine vermifuge applications on herds and the problem of residues in animal products and the environment have prompted research on the anthelmintic activity of plant extracts. This work examines the anthelmintic action of neem and the homeopathic product Fator Vermes in sheep kept in a pasture for 18 months. Forty sheep of the Morada Nova breed were divided into four treatments and the control, according to the EPG. During the experiment, each animal received 100 g/day of shredded corn and did not receive protein supplementation. In treatment 1 (control), the animals received only shredded corn. Treatment 2 received 1.6 g/(animal day) of the homeopathic product mixed with the shredded corn, and treatments 3, 4 and 5 received, respectively, 12.5, 25.0 and 37.5 g/(animal day) of dried Azadirachta indica leaves mixed with the shredded corn. The neem was administered for alternating 15-day periods and the homeopathic product daily for 18 months. There were 39 fortnightly fecal collections made to count the EPG, and fecal cultures were performed monthly. The following genera, in percentage, were identified: Haemonchus: 65.58+/-3.27, Trichostrongylus: 15.92+/-7.38 and Oesophagostomum: 18.50+/-6.22. The treatments evaluated were not effective in controlling gastrointestinal nematodes (P>0.05), whose mean log(10) counts (EPG +1) and standard errors for treatments 1-5 were respectively 3.55+/-0.28; 3.48+/-0.31; 3.90+/-0.29; 2.78+/-0.29 and 3.48+/-0.30. A significant effect (P<0.0001) was observed of the periods of the year when the 39 collections occurred. Because of the diet deficient in raw protein, the sheep had higher average EPG counts, for all the treatments, at the end of the dry season, and the opposite occurred in the middle of the rainy season.     

Occurrence of Mycobacterium spp. and other pathogens in lymph nodes of slaughtered swine and wild boars (Sus scrofa)

Mycobacterium spp. and other pathogens were investigated in 258 swine lymph nodes (129 with and 129 without apparent lesions), and 120 lymph nodes (60 with and 60 without lesions) from wild boars (Sus scrofa). A total of lymph nodes from swine and wild boars were collected of different animals. Submaxillar and mesenteric lymph nodes were submitted to microbiological examination and colonies suggestive of Mycobacterium spp. (alcohol-acid bacilli) were submitted to PCR Restriction Assay (PRA). In swine with lymphadenitis, Mycobacterium spp. (24.1%) and Rhodococcus equi (13.2%) were the most prevalent microorganisms, while in lymph nodes without lesions were identified a complex of microorganisms, including of environmental mycobacteria. In wild boars with lymphadenitis, ß-haemolytic Streptococcus (10.0%), Mycobacterium spp (8.4%) and R. equi (6.6%) were the most frequent. Among mycobacterias were identified predominantly Mycobacterium avium subspecies type 1 (48.3%) and M. avium subspecies type 2 (16.1%), followed by Mycobacterium intracellulare, Mycobacterium szulgai,Mycobacterium fortuitum, Mycobacterium gordonae, Mycobacterium simiae, Mycobacterium nonchromogenicum and Mycobacterium intracellulare type 2.     

Occurrence of weak mutators among avian pathogenic Escherichia coli (APEC) isolates causing salpingitis and peritonitis in broiler breeders

A collection of 46 avian pathogenic Escherichia coli (APEC) isolates was examined for the presence of mutators by determining the rate of mutation to rifampicin resistance. The collection included 34 E. coli isolates obtained in pure culture from chronic lesions of salpingitis and peritonitis in 34 broiler breeders, of which 12 were associated with the development of secondary septicemia. Twelve additional isolates were obtained from a clonal outbreak (ST95) of E. coli peritonitis syndrome (EPS), the lesions of which changed gradually over time into a subacute/chronic form. The hypothesis of the present study was that mutation rates would be higher for chronic infection isolates than for isolates from acute infections/exacerbations. The distribution of mutation rates followed a pattern similar to that found for other clinical isolates of E. coli, with a modal/median value of 1.47 × 10⁻⁸. Of the 46 isolates, 24% (n = 11) were weakly hypermutable (2.00 × 10⁻⁸ ≤ μ < 2.00 × 10⁻⁷), however, no strong mutators were detected (μ ≥ 2.00 × 10⁻⁷). Chronic salpingitis isolates had the highest proportion (45%, P = 0.001) of weak mutators and also, significantly higher mutation rates (P = 0.003) compared to isolates that caused septicemia (4%). In addition, mutation rates were significantly lower among ST95 isolates (P < 0.0005), and among isolates from the same clonal group as ST95 (P = 0.027), when compared to isolates from other groups. Although a clear association with the time phase of infection (as lesions of EPS became more chronic) could not be observed (ρ = 0.523, P = 0.081), a higher frequency of weak mutators among chronic infection isolates suggests that increased mutation rates play a role in adaptation of APEC to long-term persistence in an infected host environment.     

Henneguya corruscans n. sp. (Myxozoa,Myxosporea, Myxobolidae), a parasite of Pseudoplatystoma corruscans (Osteichthyes,Pimelodidae) from the Paraná River,Brazil: A morphological and morphometric study

This paper describes the parasite Henneguya corruscans n. sp. which infects the gills of Pseudoplatystoma corruscans Spix and Agassiz, 1829 found in the Paraná River, Brazil. The parasites belong to the interlamellar-epithelial type as defined by Molnár (2002) [Molnár, K., 2002. Site preference of fish myxosporeans in the gills. Dis. Aquat. Org. 48, 197–207]. The spores examined had thin, smooth walls with symmetric valves; the total length of the spores was 27.6 (25–29) μm. The spore body was ellipsoidal in frontal view and biconvex in lateral view and they measured 14.3 (13–15) μm long by 5 μm wide and 4 μm in thickness. The polar capsules were small and elongated, equally sized, with a rounded posterior extremity and tapering anteriorly, and they corresponded more or less the half the length of the spore body; they were 6.8 (6–7) μm long by 2 μm wide, and the polar filament formed 5–6 coils obliquely to the axis of the polar capsule. The tail was 13.7 (12–15) μm long and bifurcated shortly after the end of the spore body. The importance of the infection for the farming of P. corruscans is discussed.     

The prevalence and distribution of Mycobacterium bovis infection in European badgers (Meles meles) as determined by enhanced post mortem examination and bacteriological culture

The accurate diagnosis of Mycobacterium bovis infection in badgers is key to understanding the epidemiology of tuberculosis in this species and has significant implications for devising strategies to limit spread of the disease. In this study, badgers (n = 215) in the Republic of Ireland were examined at post mortem and tissues were collected from a range of anatomical locations and pooled into groups for bacterial culture of M. bovis. By assessing confirmed gross visible lesions (VL) alone, infection was detected in 12.1% of badgers. However, by including the results of all culture positive pooled samples, the overall infection prevalence increased significantly to 36.3%. Two-thirds (66.7%) of infected animals had no visible lesions (NVL). While the thoracic cavity (lungs and pulmonary lymph nodes) was found to be the most common site of infection, in a proportion of animals infection was absent from the lungs and draining lymph nodes and was confined to the lymph nodes of the carcase or the head. This may indicate an early extrapulmonary dissemination of infection or alternatively, in the case of the head lymph nodes, a secondary pathogenic pathway involving the lymphoid tissues of the upper respiratory tract (URT).     

Experimental in vitro transmission of Babesia sp. (EU1) by Ixodes ricinus

Babesia sp. (EU1), first characterized in 2003, has been implicated in human cases of babesiosis in Italy, Austria and Germany. It has been identified in roe deer and in its suspected tick vector, Ixodes ricinus, in several European countries. The aim of the present study was to validate the competence of I. ricinus as a vector of Babesia sp. (EU1) via experimental infections. For this purpose, a parasite strain isolated from roe deer was cloned in sheep erythrocytes. After experimental infections, parasite DNA was successfully amplified by PCR in both eggs and larvae originating from infected I. ricinus females and in the salivary glands of females exposed to Babesia sp. (EU1) as nymphs. We also demonstrate that infected females were able to transmit parasite DNA during a new blood meal. Together with previous epidemiological studies, these results validate I. ricinus as a competent vector for Babesia sp. (EU1).     

Expression of Mycoplasma bovis variable surface membrane proteins in the respiratory tract of calves after experimental infection with a clonal variant of Mycoplasma bovis type strain PG45

The pathomorphological findings and the expression and distribution of variable surface protein antigens (Vsp) of Mycoplasma (M.) bovis were characterised immunohistochemically in lungs of eight calves following inoculation with a Vsp A-expressing clonal variant of M. bovis type strain PG45. Within 48 h post inoculation (p.i.) an innate immune response dominated by macrophages and neutrophils develops. The monoclonal antibodies (mAbs) 1A1 and 1E5 detected M. bovis Vsp antigens in paraffin tissue sections of seven calves. Vsp antigens were widely distributed and were already present at day two p.i. within macrophages and other lung compartments. Taken together, the results demonstrate that the bovine is unable to eliminate M. bovis during the time period examined. Based on the different immunohistochemical labelling patterns obtained with the mAbs, the results also support the speculation that the in vivo variability of Vsps together with immunological factors may contribute to the chronicity of pulmonary disease.     

Sarcocystis spp. in llamas (Lama glama) in Southern Bolivia: A cross sectional study of the prevalence,risk factors and loss in income caused by carcass downgrades

Llamas (Lama glama) are intermediate hosts of the protozoan parasite Sarcocystis spp. This parasite is described as causing economic losses in the production of llama meat in South America. The aim of this study was to estimate prevalence, identify risk factors and explore spatial patterns of Sarcocystis in llamas in an area of the Bolivian High Plateau including estimating financial losses due to carcass downgrades as a result of the presence of Sarcocystis cysts.     

Mycoplasma hyopneumoniae-derived lipid-associated membrane proteins induce apoptosis in porcine alveolar macrophage via increasing nitric oxide production,oxidative stress,and caspase-3 activation

Mycoplasma hyopneumoniae is the primary etiological agent of enzootic pneumonia in swine. Lipid-associated membrane proteins (LAMP) of mycoplasma are the main pathogenicity factors in mycoplasma diseases. In this study, we investigated the effects of M. hyopneumoniae LAMP on porcine alveolar macrophage (PAM) 3D4/21 cell line. Apoptotic features, such as chromatin condensation and apoptotic bodies, were observed in LAMP-treated PAM 3D4/21 cells. Moreover, LAMP significantly increased the number of TUNEL positive apoptotic cells in PAM 3D4/21 cells compared with the untreated control. In addition, flow cytometric analysis using dual staining with annexin-V-FITC and propidium iodide (PI) showed that LAMP of M. hyopneumoniae induced a time-dependent apoptosis in PAM 3D4/21 cells. Moreover, increased levels of superoxide anion production and activated caspase-3 in PAM 3D4/21 cells were observed after exposure to LAMP. Increased production of nitric oxide (NO) was also confirmed in the cell supernatants. Besides, apoptotic rates increase and caspase-3 activation were suppressed by NOS inhibitor or antioxidant. It is suggested that LAMP of M. hyopneumoniae induced apoptosis in porcine alveolar macrophage via NO production, superoxide anion production, and caspase-3 activation.     

Cytotoxicity of Senecio in macrophages is mediated via its induction of oxidative stress

In Arunachal Pradesh and other sub-Himalayan areas of India, accidental consumption of Senecio plants by yaks is often fatal as the plant contains toxic alkaloids like Seneciophylline. The present investigation was undertaken to demonstrate the pro-oxidant effects of an ethanolic extract of Seneciochrysanthemoides (S-EtOH). S-EtOH impaired viability in macrophages, the IC₅₀ being 13.8 ± 1.11 μg/mL. The effect of S-EtOH (1 μg/mL) on generation of reactive oxygen species (ROS) in macrophages was measured by flow cytometry using 2′,7′-dichlorofluorescein diacetate (H₂DCFDA) where it caused a significant increase in the mean fluorescence channel (MFC) from 8.55 ± 0.03 to 47.32 ± 2.25 (p < 0.001). S-EtOH also effected a 3.8-fold increase in extracellular nitric oxide (NO) generation from 4.90 ± 0.72 μM to 18.79 ± 0.32 μM (p < 0.001), a 2.2-fold increase in intracellular NO production, the MFC increasing from 14.95 ± 0.48 to 33.34 ± 1.66 (p < 0.001), and concomitantly depleted non protein thiols as analyzed by flow cytometry using mercury orange, with a reduction in MFC from 632.5 ± 49.44 to 407.4 ± 12.61 (p < 0.01). Additionally, S-EtOH (14 μg/mL, 24 h) caused apoptosis as evident by increased Annexin V binding and terminal deoxynucleotidyl transferase mediated dUTP DNA nick end labeling. Taken together, the cytotoxicity of S-EtOH can be partly attributed to its capacity to inflict oxidative damage via generation of both reactive oxygen and nitrogen species culminating in apoptosis.