Use of Adipose Tissue-Derived Mesenchymal Stem Cells for Experimental Tendinitis Therapy in Equines

Superficial digital flexor tendon lesion is an important cause of lameness in equine athletes. Although numerous treatments have been described, few are effective at promoting significant improvement in the quality of the extracellular matrix. Therefore, great potential remains for recurrence and in certain cases, an abrupt end to the horse’s athletic career. Recently, several experiments have focused on the therapeutic potential of mesenchymal stem cells (MSCs) in cases of tendon lesions. This study aimed to evaluate the effect of adipose tissue-derived MSCs in the treatment of induced tendinitis of the superficial digital flexor tendon in horses by clinical, ultrasonographic, histopathological, and immunochemical analyses. Tendinitis was induced in both thoracic limbs of eight mares by administration of collagenase solution and adipose tissue was collected from the tail base for MSCs isolation and expansion, which were used during cellular therapy on only one limb 30 days after lesion induction. No differences occurred between the groups regarding the clinical and ultrasonographic analyses; however, histopathological evaluation revealed a significant improvement in tendon fiber organization and diminished inflammatory infiltrate, whereas immunohistochemical analysis showed increased expression of type I collagen in the treated group as compared with controls. The cellular therapy model implanted in this experiment promoted increased perivascular inflammatory infiltrate, fibroblastic density, neovascularization, and qualitative healing improvement of tendon extracellular matrix, in terms of fiber orientation and type I/III collagen ratio; moreover, it was considered to be a safe and viable process.     

Acetylcholinesterase cDNA sequencing and identification of mutations associated with organophosphate resistance in Cochliomyia hominivorax (Diptera: Calliphoridae)

Altered acetylcholinesterase (AChE) has been identified in numerous arthropod species resistant to organophosphate (OP) and carbamate insecticides. The New World screwworm (NWS) Cochliomyia hominivorax (Coquerel), one of the most important myiasis-causing flies in the Neotropics, has been controlled mainly by the application of OP insecticides in its current geographical distribution. However, few studies have investigated insecticide resistance in this species. Based on previous studies about mutations conferring OP resistance in related dipteran species, AChE cDNA was sequenced allowing a survey for mutations (I298V, G401A, F466Y) in NWS populations. In addition, the G137D mutation in the carboxylesterase E3 gene, also associated with OP resistance, was analyzed in the same NWS populations. Only 2/135 individuals presented an altered AChE gene (F466Y). In contrast, a high frequency of the G137D mutation in the E3 gene was found in some localities of Brazil and Uruguay, while the mutant allele was not found in Cuba, Venezuela or Colombia. These findings suggest that the alteration in the carboxylesterase E3 gene may be one of the main resistance mechanisms selected in this ectoparasite. The knowledge of the frequency of these resistance-associated mutations in the NWS natural populations may contribute to the selection of appropriate chemicals for control as part of pest management strategies.     

Anthelmintic activity of Jatropha curcas L. seeds on Haemonchus contortus

The aim of this study was to evaluate the anthelmintic activity of hexane (HE), ethyl acetate (EA) and ethanol (EE) extracts obtained from the seeds of Jatropha curcas using the egg hatch inhibition assay (EHA) and the artificial larval exsheathment inhibition assay (LEIA). For the egg hatch assay, HE, EA and EE were used in concentrations of 3.12, 6.25, 12.5, 25 and 50 mg ml(-1), accompanied by a negative control (5% Tween 80) and a positive control (0.025 g ml(-1) thiabendazole). In LEIA, the extracts were tested at a concentration of 1000 μg ml(-1), accompanied by a negative control (PBS). To evaluate the effect of tannins, the extract with the greatest effect was incubated with polyvinyl polypyrrolidone (PVPP). The EE (50 mg ml(-1)) inhibited 99.8% of egg hatching. After the addition of PVPP, the ovicidal effectiveness of EE was reduced to 91.9%. Using the HE and EA, inhibition of egg hatching was 15.3% and 32.2%, respectively. In the LEIA, 18.9% of L3 incubated with EE were exsheathed (p<0.01). The addition of PVPP to EE reversed the inhibitory effect on larval exsheathment. The percentage of exsheathment of L3 incubated with HE (99.6%) and EA (97.8%) did not differ from the control group (p>0.05). The results show that the effects of EE on eggs are not solely due to the tannins. However, these secondary metabolites are implicated in blocking the larval exsheathment.     

Assessment of in vivo and in vitro tuberculosis diagnostic tests in Mycobacterium caprae naturally infected caprine flocks

Caprine tuberculosis in Spain is mainly caused by Mycobacterium caprae although the progression of the disease and lesion severity is similar to that caused by Mycobacterium bovis. In this study, the sensitivity of the gamma-interferon (IFN-γ) assay using an antigen cocktail containing early secretory antigenic target-6kDa (ESAT-6) and culture filtrate protein 10 (CFP-10) peptides for stimulation was determined and compared with those obtained in single intradermal tuberculin (SIT) and single intradermal cervical comparative tuberculin (SICCT) tests and IFN-γ assay using purified protein derivative (PPD) in three different flocks infected with M. caprae under different epidemiological conditions. Correlation between specific IFN-γ production and severity of lesions was also evaluated. Sensitivities of the diagnostic tests varied greatly in the three flocks studied, with higher values in those where higher lesion scores were observed. The results show that IFN-γ assay applied in goats using PPD or the ESAT-6/CFP-10 peptides cocktail for stimulation yielded similar sensitivity values. A significant yet weak positive correlation between specific IFN-γ production and lesion scores was detected after the stimulation with PPDs (p=0.004) whereas when the blood samples were stimulated with ESAT-6/CFP-10 peptides, the correlation was not significant (p>0.05). Therefore, specific-IFN-γ production after the stimulation with PPDs or ESAT-6/CFP-10 was not an accurate indicator of lesion severity in naturally tuberculosis infected goats with M. caprae.     

Mycoplasma ovis in captive cervids: prevalence, molecular characterization and phylogeny

Hemotrophic mycoplasmas (hemoplasmas) are bacteria that attach to red blood cells of mammals, leading to acute and/or subclinical disease in infected animals. It has been suggested that Mycoplasma ovis, a hemoplasma that infects sheep and goats worldwide, may also infect deer. The aim of this study was to evaluate whether South American deer are infected with M. ovis. EDTA-anticoagulated blood samples from a herd of 32 captive South American deer were collected. DNA extraction of blood samples was performed followed by PCR amplification of the 16S and 23S rRNA genes, and sequencing of products. Using M. ovis PCR, 27/31 (87%) were positive, including 21/22 Mazama nana; 2/3 Mazama americana and 4/6 Blastocerus dichotomus. Sequencing of the nearly entire 16S rRNA gene of 26/27 positive samples showed 98.2-98.8% identity to M. ovis of sheep (GenBank, AF338268) and 98.6-99.4% identity to M. ovis-like of a fawn (FJ824847); the 23S rRNA gene from one of these isolates and the fawn's had 97.6% identity. The remaining isolate had just 94.9% identity to the 16S rRNA gene of M. ovis and only 89.4% identity to the 23S rRNA gene of the fawn's M. ovis. This is the first report of M. ovis in captive South American deer, revealing a high prevalence of hemoplasma infection in these animals.     

Asymptomatic encephalitis in calves experimentally infected with bovine herpesvirus-5

This study demonstrated that bovine herpesvirus 5 (BoHV)-5 infected calves can develop encephalitis and remain asymptomatic. Seven calves were infected intranasally and monitored for 30 days. Cerebrospinal fluid (CSF) analysis was performed from the onset of neurological signs. Multiple sections of brain and the trigeminal ganglion were submitted to histopathology. Virus detection (PCR and isolation) was performed on CSF and tissues. Four calves developed signs of neurologic disease and died. Three calves remained asymptomatic and were euthanized 30 days post-infection. Cerebrospinal fluid mononuclear pleocytosis occurred in symptomatic and asymptomatic calves. BoHV-5 was isolated and viral DNA was detected in multiple areas of the encephalon of all calves. The viral DNA was detected in the CSF of 2 calves showing neurological signs. Histologically, inflammation was noted in the brain of all calves and confirmed that the encephalitis caused by BoHV-5 may be mild and asymptomatic.     

Holding power of three different pin designs in the femur and ulna of the common buzzard (Buteo buteo)

External skeletal fixation is generally considered the best stabilization technique for immobilizing avian long bone fractures, but one of its major complications is the failure of bone-fixation pin interface or the loss of holding power. Consequently, this study is aimed at elucidating which pin design offers more pull-out strength in certain bones of the common buzzard (Buteo buteo). To achieve this objective, three pin designs (a smooth design and two negative profile threaded designs, with different thread pitch) were placed in five positions along the femur and ulna of the common buzzard. The pin pull-out strength was measured with the purpose of comparing medullary and pneumatic bones, insertion sites, and pin designs. Threaded pins with negative profile showed greater holding power than smooth pins (P < 0.05). When comparing holding power between the ulna and femur, no differences were found for smooth pins, whereas threaded pins showed more pull-out strength in the ulna than in the femur (P < 0.05). There were no differences observed related to pin location along the same bone when considering the same pin type. These results suggest that negative profile threaded pins have more holding power than smooth pins and that pneumatic bones provide less pull-out strength to negative profile threaded pins than medullary bones.     

Clonal population structure and introductions of the chestnut blight fungus, <Emphasis Type="Italic">Cryphonectria parasitica</Emphasis>, in Asturias,northern Spain

To understand the history of introductions of the chestnut blight fungus, Cryphonectria parasitica, in the Principality of Asturias in northern Spain, we conducted an extensive survey of chestnut blight and collected C. parasitica from 216 sites. All 778 isolates were assayed for vegetative compatibility (vc) type, whereas a subsample of 301 isolates was assayed for mating type, and 189 isolates were genotyped at 16 microsatellite markers. We found low diversity for all markers. Nearly all isolates (95%) were compatible with vc type EU-1 and had the same microsatellite multilocus haplotype, or differed from the most common type by mutation at one locus. Approximately 5% of the isolates were vegetatively compatible with EU-13 and only two isolates (< 1%) were compatible with EU-3; five different microsatellite haplotypes were found among isolates in these latter two vc types. The overall mating-type ratio was 218 MAT-1: 81 MAT-2, with both mating types represented in each of the three vc types. Microsatellite haplotypes based on ten markers used in France showed that most isolates in Asturias were either identical to or only one marker different from one of the seven most common genotypes in France, RE103. Based on these ten markers alone, the population of C. parasitica in Asturias, would appear to have been founded by a single genotype from the C1 lineage (to which RE103 belongs) found in eastern France and northern Italy. However, additional genotyping by vc types suggests the introduction of multiple genotypes, with different vc types. The exact source for introduction into Asturias cannot be determined without additional genotyping of isolates from other locations. Regardless of their origin, the low diversity of vc types makes this population ideal for deploying hypovirulence because there will be few barriers for virus transmission between individuals.     

Host-pathogen interaction of root-knot nematode <Emphasis Type="Italic">Meloidogyne incognita</Emphasis> on pepper in the southeast of Spain

The feeder roots of pepper plants (cv. California Wonder) in Campo de Cartagena (southeast Spain) were found to be severely infected by Meloidogyne incognita. Morphometric traits, differential host test and DNA analysis based on PCR were used to characterize the nematode. Naturally and artificially infected pepper plants showed severe yellowing and stunting, with heavily deformed and damaged root systems. Root galls were spherical and commonly contained more than one female and egg masses with eggs. Typical giant cells with a granular cytoplasm and many hypertrophied nuclei were observed in histological preparations. The relationship between initial nematode population density (Pi) and pepper plant growth was tested in greenhouse experiments with inoculum levels that varied from 0 to 64 eggs and second-stage juveniles (J₂) ml⁻¹ soil. A Seinhorst model was fitted to plant height and top fresh weight data of inoculated and non-inoculated plants. The tolerance limit with respect to plant height and fresh top weight of pepper to M. incognita was estimated as 0.85 eggs and J₂ ml⁻¹ soil. The minimum relative values (m) for plant height and top fresh weight were 0.15 and 0.16, respectively, at Pi ≥ 64 eggs and J₂ ml⁻¹ soil. The maximum nematode reproduction rate (Pf/Pi) was 315.4 at an initial population density (Pi) of 4 eggs and J₂ ml⁻¹ soil. The obtained results could be used as a base to establish field experiments that allow strategies to prevent surpassing the threshold of nematodes in fields that are infested.