Genotyping of Toxoplasma gondii in domestic animals from Campeche, México, reveals virulent genotypes and a recombinant ROP5 allele.

Toxoplasma gondii has at least 318 genotypes distributed worldwide, and tropical regions usually have greater genetic diversity. Campeche is a state located in the southeastern region of México and has favourable climate conditions for the replication and dissemination of this protozoan, similar to those in South American countries where broad genetic diversity has been described. Thus, in this study, 4 T. gondii isolates were obtained from tissues of stray dogs and free-range chickens in Campeche, México, and were genotyped by Mn-PCR-RFLP with 10 typing markers (SAG1, altSAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1 and Apico) and 5 virulence markers (CS3, ROP16, ROP17, ROP18 and ROP5) to provide new information about the distribution and virulence prediction of T. gondii genotypes. Two isolates of T. gondii genotype #116 and 2 of genotype #38 were obtained from stray dogs and chickens, respectively. The parasite load found in these species was between <50 and more than 35 000 tachyzoites per mg of tissue. Virulence marker genotyping revealed a recombinant 1&3 ROP5 RFLP pattern in 2 ToxoDB #116 isolates with no prediction of virulence in a murine model, while in the 2 ToxoDB #38 isolates, the ROP18/ROP5 combination predicted high virulence. Considering all the typed markers, there is a predominance of type I and III alleles, as constantly reported for the isolates characterized in various regions of México. It is crucial to determine their phenotype to corroborate the genetic virulence profile of the T. gondii isolates obtained in this study.

Immune protection induced by E2 recombinant glycoprotein of bovine viral diarrhea virus in a murine model.

Bovine viral diarrhea virus (BVDV) is considered the most important viral pathogen in ruminants worldwide due to the broad range of clinical manifestations displayed by infected animals. Therefore, infection with BVDV leads to severe economic losses in several countries' beef and dairy industries. Vaccination prevents reproductive failure and gastrointestinal and respiratory disorders caused by BVDV infection. However, considering their limitations, conventional vaccines such as live, attenuated, and killed viruses have been applied. Hence, different studies have described subunit vaccines as an effective and safe alternative for BVDV protection. Therefore, in this study, the ectodomain of E2 (E2e) glycoprotein from NADL BVDV strain was expressed in mammalian cells and used in two vaccine formulations to evaluate immunogenicity and protection against BVDV conferred in a murine model. Formulations consisted of solo E2e glycoprotein and E2e glycoprotein emulsified in adjuvant ISA 61 VG. Five groups of 6 mice of 6-to-8-week-old were immunized thrice on days 1, 15, and 30 by intraperitoneal injection with the mentioned formulations and controls. To evaluate the conferred protection against BVDV, mice were challenged six weeks after the third immunization. In addition, the humoral immune response was evaluated after vaccination and challenge. Mice groups inoculated with solo E2e and the E2e + ISA 61 VG displayed neutralizing titers; however, the E2 antibody titers in the E2e + ISA 61 VG group were significantly higher than the mice group immunized with the solo E2e glycoprotein. In addition, immunization using E2e + ISA 61 VG prevents animals from developing severe lesions in surveyed tissues. Moreover, this group acquired protection against the BVDV challenge, evidenced by a significant reduction of positive staining for BVDV antigen in the lungs, liver, and brain between the experimental groups. Our findings demonstrated that using E2e + ISA 61 VG induces greater BVDV protection by an early humoral response and reduced histopathological lesions and BVDV antigen detection in affected organs, indicating that E2e + ISA 61 VG subunit formulation can be considered as a putative vaccine candidate against BVDV. The efficacy and safety of this vaccine candidate in cattle requires further investigation.

Genotyping of toxoplasma gondii isolates from México reveals non-archetypal and potentially virulent strains for mice.

Genotyping and virulence studies of Toxoplasma gondii are essential to investigate the pathogenesis of strains circulating worldwide. In this study, eight T. gondii isolates obtained from a congenitally infected newborn, a calf, two cats, three dogs, and a wallaby from five states of México were genotyped by Mn-PCR-RFLP with 11 typing markers (SAG1, SAG2 5'3', alt. SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1 and Apico), five virulence markers (CS3, ROP16, ROP17, ROP18 and ROP5), 15 microsatellite markers (TUB-2, W35, TgM-A, B18, B17, M33, IV.1, XI.1, M48, M102, N60, N82, AA, N61, N83), and sequencing. A phylogenetic network was built to determine the relationship between Mexican isolates and those reported worldwide. Six different genotypes were identified by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), ToxoDB #8, #10, #28 (n = 3), #48, #116, and #282. Genotyping by microsatellite analysis differentiated the three PCR-RFLP genotype #28 isolates into two strains, revealing a total of seven microsatellite genotypes. Three different allele combinations of ROP18/ROP5 virulence markers were also found, 3/3, 1/1, and 4/1. The last two combinations are predicted to be highly virulent in the murine model. According to the phylogenetic network, the T. gondii strains studied here are related to archetypal strains I and III, but none are related to the strains previously reported in México. The genotypes identified in this study in different species of animals demonstrate the great genetic diversity of T. gondii in México. The ToxoDB-PCR-RFLP #28 genotype was found in three isolates from different hosts and states. Additionally, four of the isolates are predicted to be highly virulent in mice. The next step will be to perform in vitro and in vivo assays to determine the phenotype of these T. gondii isolates in murine models.

The Cysteine Protease Giardipain-1 from Giardia duodenalis Contributes to a Disruption of Intestinal Homeostasis.

In giardiasis, diarrhoea, dehydration, malabsorption, weight loss and/or chronic inflammation are indicative of epithelial barrier dysfunction. However, the pathogenesis of giardiasis is still enigmatic in many aspects. Here, we show evidence that a cysteine protease of Giardia duodenalis called giardipain-1, contributes to the pathogenesis of giardiasis induced by trophozoites of the WB strain. In an experimental system, we demonstrate that purified giardipain-1 induces apoptosis and extrusion of epithelial cells at the tips of the villi in infected jirds (Meriones unguiculatus). Moreover, jird infection with trophozoites expressing giardipain-1 resulted in intestinal epithelial damage, cellular infiltration, crypt hyperplasia, goblet cell hypertrophy and oedema. Pathological alterations were more pronounced when jirds were infected intragastrically with Giardia trophozoites that stably overexpress giardipain-1. Furthermore, Giardia colonization in jirds results in a chronic inflammation that could relate to the dysbiosis triggered by the protist. Taken together, these results reveal that giardipain-1 plays a key role in the pathogenesis of giardiasis.

Acute Disseminated Toxoplasmosis in Two Specimens of Macropus rufogriseus Caused by a Genotype so far Exclusive to South America.

Macropods are included among the species considered highly susceptible to Toxoplasma gondii infection. Clinically, it is difficult to distinguish between acute toxoplasmosis due to primary infection and reactivation of chronic latent infection in susceptible species until pathologic studies are performed. Here, we described the clinical cases and lesions found in two deceased Bennett's wallabies (Macropus rufogriseus) with a presumptive diagnosis of toxoplasmosis, as well as the genetic characterization of the T. gondii isolates obtained from these specimens. Both animals presented acute infection lesions in the lungs, liver, spleen and lymph nodes associated to T. gondii infection. Histopathology and immunohistochemistry also demonstrated tissue cysts of different sizes, indicating that the wallabies were previously infected with this parasite. Two isolates were obtained, one from each specimen and the molecular characterization was done; both isolates were the ToxoDB #116 genotype. This is the first study that reports the isolation of this particular genotype outside South America, and given the histopathological findings, it could be considered virulent for this species. The dynamics of infection that T. gondii is causing in definitive and intermediate hosts in a region allows us to know the risks to which the animals and humans that live in the area are exposed, and in the future to implement a preventive medicine plan against this parasite.

Can cloning and sequencing help to genotype positive Toxoplasma gondii clinical samples? Results and validation using SAG3 as a model.

Genotyping of T. gondii in human cases is relevant to understand the transmission patterns and epidemiology of this parasitosis. However, this genetic characterization can be hampered by the difficulty of isolating the parasite from mild or asymptomatic cases and by the detection efficiency of molecular assays such as the multilocus nested-polymerase chain reaction-restriction fragment length polymorphism (Mn-PCR-RLFP). To propose an alternative for the genotyping of positive clinical samples of T. gondii with a low amount of the parasite DNA mixed within the host DNA or mixed infections, we carried out this study to validate the sequences of the SAG3 gene of T. gondii obtained after two rounds of amplification cloned into a bacterial model, thereby achieving the separation and identification of more than one genotype of T. gondii. Also, the detection limit of the parasite DNA and the fidelity of the reagents used in the nested PCR-RFLP in artificial clinical samples by sequencing were determined. T. gondii DNA was detected from 6.25 ng of DNA and 200 parasites/mL of blood. The fidelity of the AmpliTaq Gold™ polymerase after 65 cycles of amplification was 100%. Denaturation of the products obtained after two rounds of nested PCR amplification showed no evidence of chimera or artifact production. The cloning efficiency was 97.5% (39/40 clones), and none of the experiments produced recombinant sequences. Thus, the generation of chimeras with this methodology could be ruled out. Genotyping of clinical samples is important because there is no strain selection bias, as can occur in the bioassay (where more virulent strains can be selected over nonvirulent strains), and therefore, mixed infections can be detected through cloning and sequencing. Furthermore, these two techniques could be useful tools to genotype weak amplicons of any T. gondii gene obtained during nested PCR.

SAG3 Toxoplasma gondii cloning reveals unexpected fivefold infection in the blood of feral cats in the Mexican Caribbean.

BACKGROUND: Currently, more than 300 genotypes of Toxoplasma gondii (T. gondii) have been described throughout the world, demonstrating its wide genetic diversity. The SAG3 locus is one of the genes included in the genotyping panel of this parasite. It is associated with its virulence since it participates during the invasion process of the host cells. Therefore, cloning, sequencing, and bioinformatic analysis were used to deepen the understanding of the SAG3 locus genetic diversity of T. gondii in blood samples from feral cats. RESULTS: Six different SAG3 sequences were detected, five of which were detected in one feline. Three sequences were first reported here; one of them was an intragenic recombinant. In the cladogram, four out of ten SAG3 sequences did not share nodes with others reported worldwide. CONCLUSIONS: Cloning and sequencing of samples with more than one restriction pattern by PCR-RFLP were very helpful tools to demonstrate the presence of more than three genotypes of T. gondii in the blood of feral cats from southeastern Mexico. This suggests a potential mixed infection of multiple T. gondii strains and high genetic diversity of the parasites in felines in this tropical region of Mexico.

The Extracellular Vesicles from the Commensal Staphylococcus Epidermidis ATCC12228 Strain Regulate Skin Inflammation in the Imiquimod-Induced Psoriasis Murine Model.

Extracellular vesicles (EVs) are evaginations of the cytoplasmic membrane, containing nucleic acids, proteins, lipids, enzymes, and toxins. EVs participate in various bacterial physiological processes. Staphylococcus epidermidis interacts and communicates with the host skin. S. epidermidis' EVs may have an essential role in this communication mechanism, modulating the immunological environment. This work aimed to evaluate if S. epidermidis' EVs can modulate cytokine production by keratinocytes in vitro and in vivo using the imiquimod-induced psoriasis murine model. S. epidermidis' EVs were obtained from a commensal strain (ATC12228EVs) and a clinical isolated strain (983EVs). EVs from both origins induced IL-6 expression in HaCaT keratinocyte cultures; nevertheless, 983EVs promoted a higher expression of the pro-inflammatory cytokines VEGF-A, LL37, IL-8, and IL-17F than ATCC12228EVs. Moreover, in vivo imiquimod-induced psoriatic skin treated with ATCC12228EVs reduced the characteristic psoriatic skin features, such as acanthosis and cellular infiltrate, as well as VEGF-A, IL-6, KC, IL-23, IL-17F, IL-36γ, and IL-36R expression in a more efficient manner than 983EVs; however, in contrast, Foxp3 expression did not significantly change, and IL-36 receptor antagonist (IL-36Ra) was found to be increased. Our findings showed a distinctive immunological profile induction that is dependent on the clinical or commensal EV origin in a mice model of skin-like psoriasis. Characteristically, proteomics analysis showed differences in the EVs protein content, dependent on origin of the isolated EVs. Specifically, in ATCC12228EVs, we found the proteins glutamate dehydrogenase, ornithine carbamoyltransferase, arginine deiminase, carbamate kinase, catalase, superoxide dismutase, phenol-soluble ß1/ß2 modulin, and polyglycerol phosphate α-glucosyltransferase, which could be involved in the reduction of lesions in the murine imiquimod-induced psoriasis skin. Our results show that the commensal ATCC12228EVs have a greater protective/attenuating effect on the murine imiquimod-induced psoriasis by inducing IL-36Ra expression in comparison with EVs from a clinical isolate of S. epidermidis.

Autoantibodies Against Ubiquitous and Confined Antigens in Patients With Ocular, Neuro-Ophthalmic and Congenital Cerebral Toxoplasmosis.

Toxoplasma gondii infection can trigger autoreactivity by different mechanisms. In the case of ocular toxoplasmosis, disruption of the blood-retinal barrier may cause exposure of confined retinal antigens such as recoverin. Besides, cross-reactivity can be induced by molecular mimicry of parasite antigens like HSP70, which shares 76% identity with the human ortholog. Autoreactivity can be a determining factor of clinical manifestations in the eye and in the central nervous system. We performed a prospective observational study to determine the presence of autoantibodies against recoverin and HSP70 by indirect ELISA in the serum of 65 patients with ocular, neuro-ophthalmic and congenital cerebral toxoplasmosis. We found systemic autoantibodies against recoverin and HSP70 in 33.8% and 15.6% of individuals, respectively. The presence of autoantibodies in cases of OT may be related to the severity of clinical manifestations, while in cases with CNS involvement they may have a protective role. Unexpectedly, anti-recoverin antibodies were found in patients with cerebral involvement, without ocular toxoplasmosis; therefore, we analyzed and proved cross-reactivity between recoverin and a brain antigen, hippocalcin, so the immunological phenomenon occurring in one immune-privileged organ (e.g. the central nervous system) could affect the environment of another (egg. the eye).

Toxoplasma gondii infection in European mouflons (Ovis musimon) and captive wild felines from Puebla, México.

The presence of Toxoplasma gondii in zoos is cause of alert because many susceptible species kept in captivity die of clinical toxoplasmosis. Moreover, excretion of T. gondii oocysts by infected captive wild felines into the facilities could pose a risk to workers. Herbivores in wild collections can serve as sentinels of local transmission, since they get infected by the consumption of oocysts present in ground or water. Both herbivores and felids may reveal the parasite variants which are circulating in the region. We determined the seroprevalence of T. gondii in European mouflons (n = 55) and wild felines (n = 15) from a private zoological collection located in the Eastern region of México, as well as the incidence in 41 of the mouflons using ELISA. The prevalence of T. gondii in mouflons was 14.5% (n = 55) and 17.1% (n = 41) in 2011 and 19.5% in 2012. The estimated incidence was 9.8%-12.2%. In wild felines the frequency was 80%. Four sero-positive animals (two mouflons and the two oldest African lions) were euthanized. Histopathology, conventional PCR (for B1 and SeqRep529 loci) and molecular characterization were carried out. All euthanized animals were positive to T. gondii by PCR. We identified a triple infection (I + II + III) in the brain of a mouflon. In conclusion, a high infective pressure of T. gondii in the collection was found, supported by changes in its prevalence in European mouflons. A high prevalence of infection in wild felines was determined. At least four genotypes of T. gondii are present in herbivores and carnivores, and one mouflon had a mixed infection.

Dietary Biotin Supplementation Impairs Testis Morphology and Sperm Quality.

Supplements containing pharmacological concentrations of biotin are commercially available over the counter. Classical toxicity studies have considered biotin administration as harmless; however, recent investigations have shown that biotin supplementation modifies tissue morphology without changes in toxicity markers, raising concerns about the consequences of morphological changes on tissues' functions and the safety of pharmacological concentrations of the vitamin. Testes are very sensitive to toxicants, and testicular histology is a reliable method to study its function. In this work, we investigated the effects of dietary biotin supplementation on testis morphology and spermatogenesis function using an experimental model, in which we have not observed unfavorable effects on other tissue functions or toxicity markers. Male BALB/cAnNHsd mice were fed a control or a biotin-supplemented diet (1.76 or 97.7 mg biotin/kg diet) for 8 weeks. Compared to the control group, the biotin-supplemented mice presented remarkable testis morphology changes, including increased spermatogonia layers; the cellular mechanism involved is related to increased proliferation. Sperm count and serum testosterone levels were not affected, but spermatozoa motility and morphology were significantly impaired in the biotin-supplemented mice. These results caution against the use of supplements with high concentrations of biotin and indicate that biotin's pharmacological effects on morphology need to be considered in toxicological studies.

Stray dogs in the tropical state of Chiapas, Mexico, harbour atypical and novel genotypes of Toxoplasma gondii.

Genotyping of Toxoplasma gondii remains a relevant topic of study, since genotypes can be related to the presentation and severity of toxoplasmosis. To date, 292 restriction fragment length polymorphism genotypes have been described around the world. Serosurveys in southeastern Mexico have documented exposure in over 70% of people and certain animals. Recently, we have described new genotypes and mixed infections in feral cats from Quintana Roo. Thus, the aim of this study was to genotype T. gondii and to describe its genetic variability, from naturally infected stray dogs of Chiapas, which has different geographical and climatic conditions from those found at the Yucatan Peninsula and the other parts of the country. Eleven stray dogs were captured and bled to obtain DNA, and then they were euthanized to perform necropsies and to collect target tissues. Diagnosis of T. gondii was done by quantitative real-time PCR (qPCR) and endpoint PCR. Genotyping was carried out, amplifying 12 polymorphic markers and 15 microsatellites. Atypical SAG3 gene products were cloned and sequenced. All blood samples of dogs were positive to T. gondii DNA by PCR. Two isolates were obtained from pooled heart and diaphragm tissue of two dogs. Two complete PCR-RFLP genotypes were identified (type BrIII and #28). Four animals had mixed infections. A new RFLP atypical allele for the SAG3 marker was observed; cloning and sequencing analysis of this locus revealed mixed infection by a strain identical to GT1, and one type I × II intragenic recombinant. The microsatellite analysis revealed that both isolates are atypical. Thus, atypical new genotypes of T. gondii and mixed infections were found in dogs of Chiapas. The results found here and in genotyping studies in México suggest that the southeastern region favours wide genetic diversity of T. gondii and the possible presence of virulent genotypes such as those found in central and South America.

Mixed Toxoplasma gondii infection and new genotypes in feral cats of Quintana Roo, México.

Toxoplasmosis is a zoonosis caused by Toxoplasma gondii that infects homeothermic animals, including humans. To date, as many as 287 genotypes have been described worldwide. Genetic characterization of the parasite is crucial because the parasite type can determine the presentation and severity of toxoplasmosis. Previously, we reported that the Yucatán Peninsula has a frequency of infection of over 70% in humans and other animals; moreover, there are seven species of felids, including domestic cats; thus, we hypothesized that this might be a region with a high diversity of the parasite. Nevertheless, no genotyping of this protozoan has been performed in this region. Thus, the aim of this study was to genotype T. gondii from naturally infected feral cats of Quintana Roo, within the Yucatán Peninsula, and to describe its genetic variability. Eleven feral cats were captured and bled to obtain the buffy coat; then, they were euthanized to collect target organs or tissues to extract DNA. Samples were processed by PCR for diagnosis, and ten polymorphic markers were genotyped by PCR-RFLP. Atypical GRA6 gene products were cloned and sequenced. Ten of the eleven cats were PCR positive for toxoplasmosis in blood; of these, seven had mixed infections. Also, two isolates were obtained from the heart and diaphragm of two animals. At least 23 different genotypes were detected, from which 18 are new worldwide. From the atypical GRA6 gene cloning and sequencing analysis, a mixed infection was discovered, due to one strain identical to GT1 and another to VAND. In conclusion, T. gondii genetic diversity in the region is high and different from that in other regions, with new genotypes exclusive to México and some others shared with USA and South America.

Report of an unsual case of anophthalmia and craniofacial cleft in a newborn with Toxoplasma gondii congenital infection.

BACKGROUND: We present one unusual case of anophthalmia and craniofacial cleft, probably due to congenital toxoplasmosis only. CASE PRESENTATION: A two-month-old male had a twin in utero who disappeared between the 7th and the 14th week of gestation. At birth, the baby presented anophthalmia and craniofacial cleft, and no sign compatible with genetic or exposition/deficiency problems, like the Wolf-Hirschhorn syndrome or maternal vitamin A deficiency. Congenital toxoplasmosis was confirmed by the presence of IgM abs and IgG neo-antibodies in western blot, as well as by real time PCR in blood. CMV infection was also discarded by PCR and IgM negative results. Structures suggestive of T. gondii pseudocysts were observed in a biopsy taken during the first functional/esthetic surgery. CONCLUSIONS: We conclude that this is a rare case of anophthalmia combined with craniofacial cleft due to congenital toxoplasmosis, that must be considered by physicians. This has not been reported before.

Molecular detection and sensitivity to antibiotics and bacteriocins of pathogens isolated from bovine mastitis in family dairy herds of central Mexico.

Thirty-two farms (n = 535 cows) located in the state of Guanajuato, Mexico, were sampled. Pathogens from bovine subclinical mastitis (SCM) and clinical mastitis (CLM) were identified by 16S rDNA and the sensitivity to both antibiotics and bacteriocins of Bacillus thuringiensis was tested. Forty-six milk samples were selected for their positive California Mastitis Test (CMT) (≥3) and any abnormality in the udder or milk. The frequency of SCM and CLM was 39.1% and 9.3%, respectively. Averages for test day milk yield (MY), lactation number (LN), herd size (HS), and number of days in milk (DM) were 20.6 kg, 2.8 lactations, 16.7 animals, and 164.1 days, respectively. MY was dependent on dairy herd (DH), LN, HS, and DM (P < 0.01), and correlations between udder quarters from the CMT were around 0.49 (P < 0.01). Coagulase-negative staphylococci were mainly identified, as well as Staphylococcus aureus, Streptococcus uberis, Brevibacterium stationis, B. conglomeratum, and Staphylococcus agnetis. Bacterial isolates were resistant to penicillin, clindamycin, ampicillin, and cefotaxime. Bacteriocins synthesized by Bacillus thuringiensis inhibited the growth of multiantibiotic resistance bacteria such as S. agnetis, S. equorum, Streptococcus uberis, Brevibacterium stationis, and Brachybacterium conglomeratum, but they were not active against S. sciuri, a microorganism that showed an 84% resistance to antibiotics tested in this study.

Seroprevalence and national distribution of human toxoplasmosis in Mexico: analysis of the 2000 and 2006 National Health Surveys.

Global warming has had serious implications on dispersion of infectious diseases like toxoplasmosis. Since the frequency of Toxoplasma gondii largely depends on climatic conditions, we studied its prevalence by means of 3599 samples of the National Health Survey 2000 (NHS-2000) and 2916 of the National Health and Nutrition Survey 2006 (NHNS-2006) serum banks, obtained from 1-98 year old subjects of both genders and all states of Mexico. Anti-T.gondii IgG antibodies were determined by ELISA and confirmed by western blot. Crude, epidemiologically weighted and diagnosis-performance-adjusted prevalence values were calculated. Seroprevalence changes were compared between both surveys and among regions (north, center and coast). Also, correlations between changes in temperature or humidity and those in prevalence were measured. National crude prevalence was 60.1% and 62.6% for NHS-2000 and NHNS-2006, respectively. Weighted and adjusted values were 62.5% and 40.0% for NHS-2000, and 63.7 and 43.1% for NHNS-2006. Coastal states and children presented the largest increases between surveys, while the center of the country showed a decrease. An apparently higher prevalence of T. gondii infection was observed in both surveys compared to that performed in 1987, while a geographical re-distribution was found from 2000 to 2006, with a positive correlation between temperature and frequency deltas in 21 states where prevalence increased.

Molecular diagnosis and genotyping of cases of perinatal toxoplasmosis in Mexico.

Toxoplasma gondii congenital transmission depends partially on parasite load and genotype. Both factors were examined in 4 mother/newborn pairs with perinatal infection acquired in central Mexico. Type I and type I-related strains were identified. These results add information regarding T. gondii strains prevailing in humans, although neither the genotype nor the load were related to vertical transmission or damage.

Acute toxoplasmosis in squirrel monkeys (Saimiri sciureus) in Mexico.

Toxoplasma gondii causes fatal multisystemic disease in New World primates, with respiratory failure and multifocal necrotic lesions. Although cases and outbreaks of toxoplasmosis have been described, there are few genotyping studies and none has included parasite load quantification. In this article, we describe two cases of lethal acute toxoplasmosis in squirrel monkeys (Saimiri sciureus) of Mexico city. The main pathological findings included pulmonary edema, interstitial pneumonia, hepatitis and necrotizing lymphadenitis, and structures similar to T. gondii tachyzoites observed by histopathology in these organs. Diagnosis was confirmed by immunohistochemistry, transmission electron microscopy and both end point and real time PCR. The load was between <14 and 23 parasites/mg tissue. Digestion of the SAG3 gene amplicon showed similar bands to type I reference strains. These are the first cases of toxoplasmosis in primates studied in Mexico, with clinical features similar to others reported in Israel and French Guiana, although apparently caused by a different T. gondii variant.

Patogenia de Salmonella enteritidis FT 13a y Salmonella enteritidis biovar Issatschenko en pollos de engorda / Pathogenesis of Salmonella enteritidis PT 13a and Salmonella enteritidis biovar Issatschenko in broiler chickens

The objective of this study was to determine Salmonella enteritidis phage type 13a (SE PT 13a) and Salmonella enteritidis biovar Issatschenko phage type 6a (SI) pathogenesis in 4 days old broiler chickens. Twenty-eight birds per treatment were inoculated with a dose of 1x10(8) (SE PT 13a) and 1x10(9) (SI), respectively, and fourteen chickens were inoculated with physiological saline solution (PSS) as negative controls. Samples from liver, spleen, heart, lung, crop, duodenum, jejunum, ileum and blind guts were taken during fourteen different times postinfection (6, 18, 30, 42, 54, 78, 102, 126, 150, 174, 198, 222, 246 and 270 hours postinfection (hpi) in order to obtain Salmonella spp isolation for bacteriological, histopathological and ultrastructural examination. During the first week, some depressed birds were observed, and the second week birds were found with yolk sac retention in both treatments. SE PT 13a was isolated at 18, 30, 42, 54, 78, 102, 126, 150, 174, 198, 246 and 270 hpi from all organs previously described. SI was isolated at 42, 150, 174 and 222 hpi, from crop, jejunum, ileum and blind gut samples. Ileum was the main organ where more frequently SE PT 13a and SI were isolated. There was no mortality in either treatments. Histopathology revealed inflammation, coagulative necrosis, congestion and hemorrhages in gastrointestinal tract (GIT) and visceral organs since 6 hpi in both treatments, with lesions from mild to severe. Ultrastructurally changes in enterocytes' cytoplasm: degeneration, necrosis, invasion and penetration of SE PT 13a and SI were observed. Results of this research showed the ability of SE PT 13a as well as SI to penetrate and invade enterocytes in experimentally infected broiler chicken, demonstrating that SI is able to infect broiler chickens and not only Muridae family.

El objetivo del presente estudio fue determinar la patogenia de Salmonella enteritidis fagotipo 13a (SE FT 13a) y de Salmonella enteritidis biovar Issatschenko fagotipo 6a (SI) en pollitos de engorda de cuatro días de edad. Veintiocho aves por tratamiento fueron inoculadas con dosis de 1 x 10(8) (SE FT 13a) y 1 x 10(9) (SI), respectivamente, y 14 pollitos fueron inoculados con solución salina fisiológica (SSF), como testigos negativos. Se tomaron muestras de hígado, bazo, corazón, pulmón, buche, duodeno, yeyuno, íleon y ciegos durante 14 tiempos posinfección (6, 18, 30, 42, 54, 78, 102, 126, 150, 174, 198, 222, 246 y 270 horas posinfección (hpi)), para realizar el aislamiento bacteriológico de Salmonella spp, exámenes histopatológico y ultraestructural. Durante la primera semana, se observó que algunas aves estaban deprimidas, y en la segunda semana se encontraron aves con retención de saco vitelino en ambos tratamientos. Se aisló SE FT 13a a partir de las 18, 30, 42, 54, 78, 102, 126, 150, 174, 198, 246 y 270 hpi, en todos los órganos previamente descritos. SI fue aislada a las 42, 150, 174 y 222 hpi de muestras de buche, yeyuno, íleon y ciegos. El íleon fue el órgano de donde se aisló SE FT 13a y SI con mayor frecuencia. No se registró mortalidad en ningún tratamiento. El examen histopatológico reveló inflamación, necrosis coagulativa, congestión y hemorragias en tracto gastrointestinal (TGI) y órganos viscerales a partir de las 6 hpi en ambos tratamientos, con grados de lesión de leve a severo. Ultraestructuralmente se observaron cambios en el citoplasma celular de enterocitos: degeneración, necrosis, invasión y penetración de SE FT 13a y SI. Los resultados de la presente investigación evidenciaron la capacidad de SE FT 13a y SI para penetrar e invadir enterocitos de los pollitos infectados experimentalmente, demostrando que SI es capaz de infectar pollos de engorda y no sólo a la familia Muridae.

Casiopeina III-ia induces apoptosis in HCT-15 cells in vitro through caspase-dependent mechanisms and has antitumor effect in vivo.

The aim of this study was to evaluate the in vitro and in vivo effects of the new chemotherapy agent Casiopeina III-ia [(4,4'-dimethyl-2,2'-bipiridine)(acetylacetonate) Copper (II) nitrate] on HCT-15 (p53-/-) colon cellular line. In vitro, the drug reduced the viability and induced necrosis and apoptosis in a dose dependent manner, without affecting cell cycle phases. Apoptosis was related to Bax increasing levels, suggesting a caspase-dependent mechanism of death, as verified by nucleosomal fragmentation of DNA. In vivo, the antitumor activity of Casiopeina III-ia was tested in HCT-15 cells transplanted to nude mice. In this study we will show that the novel antineoplastic agent Casiopeina III-ia is active on this colon tumor line, setting out as a good candidate for the treatment of colon tumors refractory to chemotherapy.