Novel Gluconobacter oxydans strains selected from Kombucha with potential postbiotic activity.

Gastric and colorectal cancer are among the most frequently diagnosed malignancies of the gastrointestinal tract. Searching for methods of therapy that complements treatment or has a preventive effect is desirable. Bacterial metabolites safe for human health, which have postbiotic effect, are of interest recently. The study aimed to preliminary assessment of the safety, antimicrobial, and anti-cancer activity of cell-free metabolites of Gluconobacter oxydans strains isolated from Kombucha beverages as an example of the potential postbiotic activity of acetic acid bacteria (AAB). The study material consisted of five AAB strains of Kombucha origin and three human cell lines (gastric adenoma-AGS, colorectal adenoma-HT-29, and healthy cells derived from the endothelium of the human umbilical vein-HUVEC). Results of the study confirms the health safety and functional properties of selected AAB strains, including their potential postbiotic properties. The best potential anticancer activity of the AAB cell-free supernatants was demonstrated against AGS gastric adenoma cells. The conducted research proves the postbiotic potential of selected acetic acid bacteria, especially the KNS30 strain. KEY POINTS: •The beneficial and application properties of acetic acid bacteria are poorly studied. •Gluconobacter oxydans from Kombucha show a postbiotic activity. •The best anticancer activity of the G. oxydans showed against gastric adenoma.

Identification of intestinal parasites in wild American mink (Neovison vison) from Biebrza and Narew national parks (Poland).

American mink (Neovison vison) is an invasive species in the sylvatic environment of Poland. Mink are exposed to different parasite infections as their preys serve as intermediate and/or paratenic hosts. The study aimed to discriminate the pattern of intestinal parasite infections in mink inhabiting Biebrza (BNP) and Narew (NNP) national parks. Gastrointestinal tract examinations revealed Coccidia, Echinostomatidae, Taenidae, and Capillariidae parasites. There was no significant difference in the parasite burden of mink, but patterns of infections varied between both localizations. Coccidia were found in 3.8% of BNP vs. 6.7% of NNP mink. Fluke prevalence was significantly higher in NNP 27.5% compared to 7.7% in BNP mink. Tapeworms were only found in 3.4% of NNP mink. Significantly more Aonchotheca eggs were found in BNP 34.6% vs. 11.4% in NNP mink. The intensity of coccidiosis and aonchothecosis was low in both parks. Fluke intensity varied between low to moderate (ranging from 1 to 16) in BNP and low to massive (ranging from 1 to 117) in NNP mink. Coinfections of various parasite species were noted in both areas. Morphological and DNA analysis revealed that flukes belonged to Isthiomorpha melis and tapeworms to Versteria mustelae. It was the first isolation of V. mustelae in mink of those localizations. In conclusion, our study showed that mink indwelling Biebrza and Narew national parks are moderately infested with parasites. Results suggest that mink play an important role as a reservoir for parasites endangering endemic mustelids, becoming also a potential risk factor in case of accidental transmissions to farm mink. That is why, more strict biosecurity measures are required to protect farm mink.

Cytokine production and signalling in human THP-1 macrophages is dependent on Toxocara canis glycans.

The effect of Toxocara canis antigens on cytokine production by human THP-1 macrophages was studied in vitro. Toxocara Excretory-Secretory products (TES) and recombinant mucins (Tc-MUC-2, Tc-MUC-3, Tc-MUC-4, and Tc-MUC-5) as well as deglycosylated forms of these antigens were used in the study. TES products stimulated macrophages to produce the innate proinflammatory IL-1ß, IL-6, and TNF-α cytokines regardless of the presence of glycans. Recombinant mucins induced glycan-dependent cytokine production. Sugar moieties led to at least 3-fold higher production of regulatory IL-10 as well as proinflammatory cytokines. The presence of glycans on mucins also affected the downstream signalling pathways in stimulated cells. The most prominent difference was noted in AKT and AMPK kinase activation. AKT phosphorylation was observed in cells stimulated with glycosylated mucins, whereas treatment with deglycosylated antigens led to AMPK phosphorylation. MAP kinase family members such as JNK and p38 and c-Jun transcription factor were phosphorylated in both cases what suggests that toll-like receptor signalling may be involved in mucin-treated macrophages. This pathway is however modified by other signalling molecules as only mucins containing intact sugars significantly induced the production of cytokines.

Hamsters vaccinated with Ace-mep-7 DNA vaccine produced protective immunity against Ancylostoma ceylanicum infection.

Hookworms are intestinal nematodes that infect up to 740 million people, mostly in tropical and subtropical regions. Adult worms suck blood from damaged vessels in the gut mucosa, digesting hemoglobin using aspartic-, cysteine- and metalloproteases. Targeting aspartic hemoglobinases using drugs or vaccines is therefore a promising approach to ancylostomiasis control. Based on homology to metalloproteases from other hookworm species, we cloned the Ancylostoma ceylanicum metalloprotease 7 cDNA (Ace-mep-7). The corresponding Ace-MEP-7 protein has a predicted molecular mass of 98.8 kDa. The homology to metallopeptidases from other hookworm species and its predicted transmembrane region support the hypothesis that Ace-MEP-7 may be involved in hemoglobin digestion in the hookworm gastrointestinal tract, especially that our analyses show expression of Ace-mep-7 in the adult stage of the parasite. Immunization of Syrian golden hamsters with Ace-mep-7 cDNA resulted in 50% (p < 0.01) intestinal worm burden reduction. Additionally 78% (p < 0.05) egg count reduction in both sexes was observed. These results suggest that immunization with Ace-mep-7 may contribute to reduction in egg count released into the environment during the A. ceylanicum infection.

Toxocara canis mucins among other excretory-secretory antigens induce in vitro secretion of cytokines by mouse splenocytes.

The effect of Toxocara larval antigens on cytokine secretion by mouse splenocytes was studied in vitro. Recombinant mucins were produced in Pichia pastoris yeast, and Toxocara excretory-secretory (TES) antigens were collected from in vitro culture of L2 larvae. Tc-MUC-2, Tc-MUC-3, Tc-MUC-4, and Tc-MUC-5 were expressed as glycoproteins and were specifically recognized by Toxocara canis-infected dog serum antibodies. Mouse splenocytes stimulated with recombinant mucins produced IL-5, IL-6, and TGF-ß. Cell stimulation with whole TES products was more effective and resulted in secretion of IL-4, IL-5, IL-6, IL-10, and TGF-ß and downregulation of TNF-α production. IFN-γ and IL-17 secretion was noted only after ConA treatment. Cells originating from infected animals produced significantly smaller amounts of these two cytokines compared to control cells, which suggests that Th1 and Th17 response in infected mice is strongly inhibited. However, splenocyte stimulation with both TES and ConA upregulated the production of IFN-γ and IL-17. This shows that TES antigens have strong immunomodulatory properties and are able to induce a broad range of effects on murine immune cells.

Increased concentration of serum TNF alpha and its correlations with arterial blood pressure and indices of renal damage in dogs infected with Babesia canis.

Canine babesiosis is a tick-borne disease caused by parasites of the genus Babesia. Tumour necrosis factor alpha (TNF-α) is a cytokine that plays a role in the pathogenesis of canine babesiosis. In this study, the authors determined the concentration of serum TNF-α in 11 dogs infected with Babesia canis and calculated Spearman's rank correlations between the concentration of TNF-α and blood pressure, and between TNF-α and indices of renal damage such as: fractional excretion of sodium (FE(Na(+))), urinary creatinine to serum creatinine ratio (UCr/SCr), renal failure index (RFI), urine specific gravity (USG) and urinary protein to urinary creatinine ratio (UPC). The results demonstrated statistically significant strong negative correlations between TNF-α and systolic arterial pressure (r = -0.7246), diastolic arterial pressure (r = -0.6642) and mean arterial pressure (r = -0.7151). Serum TNF-α concentration was also statistically significantly correlated with FE(Na(+)) (r = 0.7056), UCr/SCr (r = -0.8199), USG (r = -0.8075) and duration of the disease (r = 0.6767). The results of this study show there is an increase of serum TNF-α concentration during canine babesiosis, and the increased TNF-α concentration has an influence on the development of hypotension and renal failure in canine babesiosis. This probably results from the fact that TNF-α is involved in the production of nitric oxide and induction of vasodilation and hypotension, which may cause renal ischaemia and hypoxia, and finally acute tubular necrosis and renal failure.

Analysis of the role of Dirofilaria repens macrophage migration inhibitory factors in host-parasite interactions.

Introduction: Dirofilaria repens is a zoonotic parasitic filarial nematode that infects carnivores and occasionally humans. Knowledge of the host-parasite molecular interactions enabling the parasite's avoidance of the host immune response in subcutaneous dirofilariasis remains limited. Parasitic orthologues of host macrophage migration inhibitory factor (MIF) are molecules potentially involved in this process. Material and Methods: Complementary DNA encoding two D. repens MIF orthologues (rDre-MIF-1 and rDre-MIF-2) was cloned into a pET-28a expression vector. The recombinant proteins were produced in Escherichia coli and purified using affinity nickel chromatography. The reactivity of both recombinant proteins was analysed with infected dog and immunised mouse sera. Results: Stronger antibody production was induced by rDre-MIF-1 in mice, as evidenced by significantly higher levels of anti-rDre-MIF-1 total IgG, IgG2 and IgE antibodies than of anti-rDre-MIF-2 immunoglobulins. Additionally, a significantly different level of antibodies specific to both proteins was noted between the sera of infected dogs and those of uninfected dogs. Conclusion: This study is the first attempt to characterise MIF orthologues from the filarial parasite D. repens, which may affect the immune response during infection.

Molecular cloning and characterisation of in vitro immune response against astacin-like metalloprotease Ace-MTP-2 from Ancylostoma ceylanicum.

Ancylostoma ceylanicum belongs to the group of parasites commonly known as hookworms, blood-sucking nematodes which infect around 576 million people and hundreds of millions of animals. The interactions between these parasites and host immune systems are complicated and yet to be determined. Hookworm infections are usually long lasting and recurrent, due in part to their ability to synthesize macromolecules capable of modulating the host immune response. The interaction of parasite proteins with host immune systems has been proven, but so far there is no data describing the influence of astacin-like metalloproteases (expressed among different parasitic nematodes) on the human immune system. The cDNA encoding A. ceylanicum metalloprotease 2 (Ace-mtp-2) was cloned using RACE-PCR. Computational analysis was used to examine the immunogenicity and recombinant Ace-MTP-2 was used to investigate its influence on human THP-1 monocytes and macrophages. The Ace-mtp-2 gene encodes an astascin-like metalloprotease, with a theoretical molecular mass of 26.7 kDa. The protease has a putative signal peptide, 11 potential phosphorylation sites, and two disulfide bridges revealed by computational analysis. Maximal expression of Ace-mtp-2 by A. ceylanicum occurs in the adult stage of the parasite, and Western blot indicates the secretory nature of the protease. This suggests the protease is working at the host-parasite interface and would likely be exposed to the hosts immune response. Recombinant protein were expressed in Escherichia coli and Pichia pastoris. Recombinant Ace-MTP-2 amplified the in vitro release of TNFα and induced release of IFNγ by lipopolysaccharide activated THP-1 macrophages. The presence of Ace-MTP-2 in secretory products of the adult parasite and the induction of IFNγ release may suggest an important role for Ace-MTP-2 in host-parasite interactions since IFNγ is suggested to be responsible for the protective immune response against adult hookworms.

Immune Response of the Host and Vaccine Development.

Vaccines are one of the greatest achievements of modern medicine, offering an effective way to fight and control infectious diseases [...].

Canine antibody response against Dirofilaria repens in natural occult and microfilaremic infections.

Dirofilaria repens is the causative agent of the zoonotic canine skin condition called subcutaneous dirofilariosis. Despite being endemic throughout much of Europe, little is known about host humoral responses to D. repens. To address this, we analyzed serum immunoglobulin isotypes recognizing D. repens somatic antigens (DrSA) in naturally infected dogs. Titers of anti-DrSA IgG and IgE, but not IgM were significantly higher in dogs infected with D. repens compared to those that were negative. Moreover, microfilaremic infections were associated with higher levels of IgG1 than IgG2, while occult infections occurred with significantly higher levels of IgG2 than IgG1. Finally, the measurement of anti-DrSA IgG antibodies allowed the detection of occult subcutaneous dirofilariosis in dogs.

The Immunological Role of Vascular and Lymphatic Endothelial Cells in Filarial Infections.

The embryonic stage of filarial nematodes, or microfilariae (Mf), shows daily and seasonal periodicity that requires their migration through blood vessels into the lungs, where they are sequestered when not circulating in the peripheral blood. Therefore, Mf and the host endothelium are likely in a permanent state of hide and seek. Interestingly, filarial nematodes co-cultured in media with a murine endothelial cell line survive eight times longer than those cultured in media alone. This suggests that the endothelium is an important element of the immune response in filarial nematodes, perversely promoting their survival in the host. In this review, we will focus on potential pathways involved in the relationship between filarial nematodes and the host endothelium, including the role of endothelial ICAM/VCAM/PECAM adhesion molecules, surface markers involved in the passage of Mf through host tissue, anti-thrombolic effects caused by the presence of filarial nematodes (including plasmins), endothelial cell proliferation (VEGF), and other aspects of the immune activation of the endothelium. The aim of this review is to merge the knowledge about the cross-talk between Mf of different filarial nematode species and endothelial cells (EC), thus allowing a better understanding of the mechanism of these parasitic infections.

Nematode Orthologs of Macrophage Migration Inhibitory Factor (MIF) as Modulators of the Host Immune Response and Potential Therapeutic Targets.

One of the adaptations of nematodes, which allows long-term survival in the host, is the production of proteins with immunomodulatory properties. The parasites secrete numerous homologs of human immune mediators, such as macrophage migration inhibitory factor (MIF), which is a substantial regulator of the inflammatory immune response. Homologs of mammalian MIF have been recognized in many species of nematode parasites, but their role has not been fully understood. The application of molecular biology and genetic engineering methods, including the production of recombinant proteins, has enabled better characterization of their structure and properties. This review provides insight into the current state of knowledge on MIF homologs produced by nematodes, as well as their structure, enzymatic activity, tissue expression pattern, impact on the host immune system, and potential use in the treatment of parasitic, inflammatory, and autoimmune diseases.

Selection of new diagnostic markers for Dirofilaria repens infections with the use of phage display technology.

Dirofilaria repens is a parasitic nematode causing vector-borne disease (dirofilariasis), considered an emerging problem in veterinary and human medicine. Although main hosts are carnivores, particularly dogs, D. repens shows high zoonotic potential. The disease spreads uncontrollably, affecting new areas. Since there is no vaccine against dirofilariasis, the only way to limit disease transmission is an early diagnosis. Currently, diagnosis depends on the detection of microfilariae in the host bloodstream using modified Knott's test or multiplex PCR. However, the efficacy of tests relying on microfilariae detection is limited by microfilariae periodic occurrence. Therefore, a new reliable diagnostic test is required. Our study aimed to select new diagnostic markers for dirofilariasis with potential application in diagnostics. We focused on single epitopes to ensure high specificity of diagnosis and avoid cross-reactivity with the other parasite infections common in dogs. Using phage display technology and 12-mer peptides library, we selected epitopes highly reactive with IgG from sera of infected dogs. Additionally, our study presents the possibility of detecting D. repens specific cell-free DNA in dogs with no microfilaria but high IgG and IgM antibody levels against parasite somatic antigen.

Identification of Toxocara canis Antigen-Interacting Partners by Yeast Two-Hybrid Assay and a Putative Mechanism of These Host-Parasite Interactions.

Toxocara canis is a zoonotic roundworm that infects humans and dogs all over the world. Upon infection, larvae migrate to various tissues leading to different clinical syndromes. The host-parasite interactions underlying the process of infection remain poorly understood. Here, we describe the application of a yeast two-hybrid assay to screen a human cDNA library and analyse the interactome of T. canis larval molecules. Our data identifies 16 human proteins that putatively interact with the parasite. These molecules were associated with major biological processes, such as protein processing, transport, cellular component organisation, immune response and cell signalling. Some of these identified interactions are associated with the development of a Th2 response, neutrophil activity and signalling in immune cells. Other interactions may be linked to neurodegenerative processes observed during neurotoxocariasis, and some are associated with lung pathology found in infected hosts. Our results should open new areas of research and provide further data to enable a better understanding of this complex and underestimated disease.

A review of the occurrence and clinical consequences of protozoan infections in carnivorous fur farm animals.

INTRODUCTION AND OBJECTIVE: The aim was to review available data concerning the occurrence of protozoan parasites affecting carnivorous fur farm animals, such as: American mink, blue and silver foxes, and raccoon dogs. Although, many protozoan pathogens have been isolated in wild fur animals, some are still not recognised as relevant to particular fur farm animal species. Protozoans that have been isolated as causative agents of clinical cases and reported in these animals are: Cryptosporidium spp., Eimeria spp., Isospora spp., Leishmania infantum, Neospora caninum, Sarcocystis spp., and Toxoplasma gondii. Unfortunately, in most reports, neither clinical symptoms nor detailed pathogenicity and clinical pathology are satisfactory discriminated. This data is essential for preparing treatment and preventive measure protocols. Additionally, attempts to better understand the infections might be useful in the case of outbreaks. RESULTS: The results of the literature research revealed that some detected infections may have zoonotic potential, and are quite likely to be transmitted in both directions. The role of vectors, e.g. small rodents, birds, insects (sand-flies), or pets (cats and dogs), which might be involved in the transmission of various parasitic infections, is also discussed. The summarized list of protozoans involved can be used for further studies on the health and welfare aspects of fur farm animal breeding and public health issues.

Molecular identification of tapeworms from Konik Polski horses from Biebrza National Park.

Tapeworm infections in Konik Polski horses from Biebrza National Park were investigated in this study. Faecal samples were collected 10 times: in 2012 - 1 time, in 2013 - 4 times, in 2014 - 4 times and in 2015 - 1 time. In total, 162 faecal samples were collected and tested. Faecal egg counts (FECs) method was used in the study. Positive samples with cestode eggs were noted only twice - in October 2012 and December 2013 in two adult mares (9 and 11 years old). The determined prevalence was surprisingly low comparing to other studies, 4.3% in October 2013 and 28.5% in December 2013. Parasite genomic DNA was isolated from posterior proglottids of tapeworms found in horse faeces after deworming, PCR technique was applied in order to determine the species of these tapeworms. No specific products were obtained using primers specific to rDNA sequence, whereas, a 620 bp fragment encoding mitochondrial cytochrome oxidase I (COI) was amplified and sequenced. Nucleotide sequence analysis revealed a 100% homology to a corresponding fragment form Anoplocephala perfoliata tapeworm originating from a horse from the Czech Republic. To our knowledge this is the first report applying molecular techniques to tapeworm identification in Konik Polski horses.

Growth and adhesion inhibition of pathogenic bacteria by live and heat-killed food-origin Lactobacillus strains or their supernatants.

The study aimed to evaluate qualitatively and quantitatively the antimicrobial capacity of 10 potential probiotic Lactobacillus strains against model enteropathogens and spoilage microorganisms. The probiotic strains (live and heat-killed forms) were also assessed for their ability to inhibit adhesion of selected pathogens to Caco-2 cells. The largest inhibition zones (the diffusion method) were connected with the usage of whole bacteria cultures (WBC), also high and moderate with cell-free supernatant (CFS) and the lowest with cell-free neutralized supernatant (CNS). The highest antagonistic activity of Lactobacillus strains was observed against L. monocytogenes strains, moderate activity against Salmonella, Shigella, Escherichia coli, Pseudomonas and, the lowest against S.aureus, Bacillus and Enterococcus. The inhibition of adhesion to Caco-2 cells was very high in the case of E. coli, Salmonella and L. monocytogenes, and moderate in the case of S.aureus. On average, the inhibition effect was higher when pathogenic bacteria were treated by WBC, than heat-killed Lactobacillus. Although, in most samples, the effect was not significantly different (P> 0.05). The strains Lb. brevis O24 and Lb. rhamnosus K3 showed the biggest overall antimicrobial properties, and were most effective in adherence inhibition of investigated indicator strains. These bacteria or their metabolites can be used for the production of various foods or pharmaceutical products.

Detection of pathogens in ixodid ticks collected from animals and vegetation in five regions of Ukraine.

The distribution and prevalence of zoonotic pathogens infecting ixodid ticks in Western Europe have been extensively examined. However, data on ticks and tick-borne pathogens in Eastern Europe, particularly Ukraine are scarce. The objective of the current study was, therefore, to investigate the prevalence of Anaplasma phagocytophilum, Anaplasmataceae, Rickettsia spp., Babesia spp., Bartonella spp., and Borrelia burgdorferi sensu lato in engorged and questing ixodid ticks collected from five administrative regions (oblasts) of Ukraine, namely Chernivtsi, Khmelnytskyi, Kyiv, Ternopil, and Vinnytsia. The ticks were collected from both wild and domestic animals and from vegetation. Of 524 ixodid ticks collected, 3, 99, and 422 ticks were identified as Ixodes hexagonus, Ixodes ricinus, and Dermacentor reticulatus, respectively. DNA samples individually extracted from 168 questing and 354 engorged adult ticks were subjected to pathogen-specific PCR analyses. The mean prevalence in I. ricinus and D. reticulatus were, respectively: 10 % (10/97) and 3 % (12/422) for A. phagocytophilum; 69 % (67/97) and 52 % (220/422) for members of the Anaplasmataceae family; 25 % (24/97) and 28 % (117/422) for Rickettsia spp.; 3 % (3/97) and 1 % (6/422) for Babesia spp.; and 9 % (9/97) and 5 % (20/422) for Bartonella spp. Overall, between the five cities, there was no significant difference in the prevalence of any of the pathogens for the respective ticks (p > 0.05). The prevalence of B. burgdorferi s. l. in the questing and engorged I. ricinus varied from 0 to 27 % and 14-44%, respectively, with no statistical significance identified between the five cities (p > 0.05). In addition to reporting the updated data for Kyiv and Ternopil, this study is the first to provide the prevalences of the tick-borne pathogens for Chernivtsi, Khmelnytskyi, and Vinnytsia. This investigation is also the first to detect Neoehrlichia mikurensis in ixodid ticks from Ukraine. These new data will be useful for medical and veterinary practitioners as well as public health officials when diagnosing infections and when implementing measures to combat tick-borne diseases in Ukraine.

Lactobacillus plantarum Strains Isolated from Polish Regional Cheeses Exhibit Anti-Staphylococcal Activity and Selected Probiotic Properties.

Twenty-nine Lactobacillus plantarum strains isolated from different types of Polish regional cheeses (Oscypek and Korycinski) were assessed for selected probiotic properties and anti-staphylococcal activity. Most of the tested L. plantarum strains were considered safe. Whole bacterial cultures (WBC) and cell-free supernatants (CFSs) of L. plantarum strains inhibited growth of Staphylococcus aureus (average inhibition growth zones were 2.8 mm ± 1.2 and 2.8 mm ± 1.1 respectively). Moreover, almost all neutralized, catalase-treated cell-free supernatants (CFN) of L. plantarum cultures also exhibited slight anti-staphylococcal activity in vitro. The most promising strains Os4 and Kor14 were selected for further study. Both strains were able to survive during digestive gastro-intestinal passage model. Live cells of L. plantarum Os4 and Kor14 caused the strongest inhibition of S. aureus adhesion to Caco-2 cells comparing with CFN and heat-killed bacterial cells. S. aureus and L. plantarum (Os4 or Kor14) co-cultured in skim milk resulted in growth inhibition of S. aureus in both 8 °C and 37 °C incubation temperatures. Observed abilities, demonstrated for L. plantarum Os4 and Kor14, confirms that these strains could be used in the food industry as protective cultures.

Case Studies of Severe Microfilaremia in Four Dogs Naturally Infected With Dirofilaria repens as the Primary Disease or a Disease Complicating Factor.

Subcutaneous dirofilariosis in dogs, caused by Dirofilaria repens, is an underdiagnosed disease, now recognized for its zoonotic potential, and growing distribution and prevalence across Europe and Asia. Our understanding of the pathogenicity in human and canine host remains unclear, but case reports suggest that microfilariae (Mf) as well as adult D. repens may directly cause internal organs damage or may be a factor complicating the course of other ailments. The purpose of the study was to report high Mf in dogs and to discuss potential relevance with co-morbidity. Our data from a modified Knott's test performed on 62 infected dogs indicate that the median Mf count in D. repens infections is 675 Mf/ml and we consider microfilaremia above 10,000 Mf/ml as high intensity. This collection of case reports discusses 4 cases of high intensity D. repens microfilaremia in companion dogs; one presenting pathology from a very high intensity of adult D. repens with post-treatment complications, and 3 dogs in which high microfilaremia was detected incidentally during the management of other primary illnesses. To our knowledge this report describes the highest D. repens microfilaremia ever detected in a dog, at 178,000 Mf/ml. The issue of high microfilaremic infections in dogs is poorly studied and there is growing need to identify the presentation and understand the mechanisms of associated pathogenesis in the host-parasite relationship.