Molecular typing and antimicrobial resistance profiling of 33 mastitis-related Staphylococcus aureus isolates from cows in the Comarca Lagunera region of Mexico.

Mastitis in cows is a major cause of economic losses and it is commonly associated with Staphylococcus aureus. Little is known about the S. aureus lineages causing mastitis in Mexican cattle. The aim of this study was to type S. aureus isolates causing mastitis in cows from the Comarca Lagunera region in Mexico in 2015-2016. Multi-locus variable number tandem repeat fingerprinting (MLVF) of 33 S. aureus isolates obtained from 210 milk samples revealed the MLVF clusters A (n = 1), B (n = 26), C (n = 5) and D (n = 1). Spa-typing showed that clusters A and B represent the spa-type t224, cluster C includes spa-types t3196 and t416, and cluster D represents spa-type t114. The different spa-types were mirrored by the masses of protein A bands as detected by Western blotting. Antimicrobial susceptibility testing showed that one isolate was susceptible to all antimicrobials tested, whereas all other strains were resistant only to benzylpenicillin. These findings show that only four S. aureus lineages, susceptible to most antimicrobials, were responsible for causing mastitis at the time of sampling. Lastly, many isolates carried the same small plasmid, designated pSAM1. The high prevalence of pSAM1 amongst the antimicrobial-susceptible isolates suggests an association with bovine colonization or mastitis rather than antimicrobial resistance.

Molecular characterisation of Porcine rubulavirus (PorPV) isolates from different outbreaks in Mexico.

Since the report of the initial outbreak of Porcine rubulavirus (PorPV) infection in pigs, only one full-length genome from 1984 (PorPV-LPMV/1984) has been characterised. To investigate the overall genetic variation, full-length gene nucleotide sequences of current PorPV isolates were obtained from different clinical cases of infected swine. Genome organisation and sequence analysis of the encoded proteins (NP, P, F, M, HN and L) revealed high sequence conservation of the NP protein and the expression of the P and V proteins in all PorPV isolates. The V protein of one isolate displayed a mutation that has been implicated to antagonise the antiviral immune responses of the host. The M protein indicated a variation in a short region that could affect the electrostatic charge and the interaction with the membrane. One PorPV isolate recovered from the lungs showed a mutation at the cleavage site (HRKKR) of the F protein that could represent an important factor to determine the tissue tropism and pathogenicity of this virus. The HN protein showed high sequence identity through the years (up to 2013). Additionally, a number of sequence motifs of very high amino acid conservation among the PorPV isolates important for polymerase activity of the L protein have been identified. In summary, genetic comparisons and phylogenetic analyses indicated that three different genetic variants of PorPV are currently spreading within the swine population, and a new generation of circulating virus with different characteristics has begun to emerge.

Long-term RNA persistence of porcine rubulavirus (PorPV-LPMV) after an outbreak of a natural infection: the detection of viral mRNA in sentinel pigs suggests viral transmission.

The persistence of porcine rubulavirus (PorPV-LPMV) in five pigs that had survived an outbreak of a natural infection was determined. After the resolution of the outbreak, each animal was housed in an isolation pen together with one sentinel pig. Approximately every 2 months thereafter one group of animals was euthanized and tissue samples taken for virological and serological analysis. Infectious virus was not isolated from any samples; antibodies to PorPV-LPMV were detected in convalescent pigs by virus neutralisation test and blocking ELISA but not in sentinel pigs. PorPV-LPMV mRNA of the nucleoprotein (NP) and phosphoprotein (P) genes was detected by a nested polymerase chain reaction (nPCR) in samples of trigeminal and optic nerves, cervical spinal cord, tonsils, salivary gland, lung and pancreas from convalescent pigs. mRNA was also detected in the midbrain, corpus callosum, or olfactory bulb in four out of five pigs by nRT-PCR, this result was confirmed by the sequencing of a 260bp PCR product of P gene region. The highest average viral copies/µg of total RNA occurred in the olfactory bulb and pancreas tissues of convalescent pigs and midbrain, tonsil and pancreas of sentinel pigs housed with the convalescent pigs. Satellitosis and gliosis of the midbrain, olfactory bulb, corpus callosum, medulla oblongata or choroid plexus were microscopically observed in four convalescent pigs. The control pig remained negative in all tests. The results indicate that PorPV-LPMV mRNA persists and induces a durable humoral immune response in pigs that have recovered from a natural infection. After a possible reactivation of the virus, it was transmitted to sentinel pigs in contact with the convalescent pigs.

The porcine circovirus type 1 in porcine kidney 15 cell line is not transferred to mice lymphoid cells after xenoimplantation into the peritoneal cavity.

The porcine circovirus type 1 (PCV1) has been identified within lymphoid tissues of experimental infected pigs and suggested to induce an immunosuppressive stage in pigs. The virus does not induce a cytophatic effect in the pig-derived cell line PK-15. Because PCV1 is prevalent in many pig cells and tissues, the risk of inducing a viral xenozoonosis by PCV1 was raised for the xenoimplantation of pig cells into human hosts. The present work evaluated if PCV1 is able to replicate in mice tissues after xenoimplantation of PCV1-infected pig cells. Active growing PK-15 cells harboring PCV1 with or without microencapsulation in sodium alginate were implanted into the peritoneal cavity of mice. After 1 month postimplantation in mice, peritoneal macrophages, spleen, and lymph nodes were harvested and analyzed with the polymerase chain reaction technique (PCR). No evidence of circovirus type 1 DNA was detected within the mice tissues.

Chorionic gonadotropin hormone receptors on Taenia solium and Taenia crassiceps cysticerci in culture.

Hormones play a significant role in murine Taenia crassiceps cysticercosis, and they may also participate in the susceptibility to Taenia solium cysticercosis. In the present study, in vitro effects are reported for human chorionic gonadotropin (hCG) on the larval stages of T. crassiceps (WFU strain) and T. solium. Our results reveal the presence of receptors for hCG in different developmental phases of both cultured parasites. On day 30, both taeniid species had the highest percentage of receptors in the neck, strobila, and suckers, but these receptors decreased by day 60, delimiting the segments and the exterior of the developing proglottids in T. solium. At the same time, there was a large number of hCG receptors in the area of the presumptive cirrus organ and in calcareous corpuscles within the parenchyma. This is the first report detecting receptors for hCG on different larval stages of T. crassiceps and T. solium. A direct effect of hCG could be recognized by the cysticerci as a factor contributing to the growth and development of T. crassiceps and T. solium cysticerci, respectively.

Experimental porcine rubulavirus (La Piedad-Michoacan virus) infection in pregnant gilts.

Porcine rubulavirus (La Piedad-Michoacan virus) (PoRV-LPMV) is a member of the Paramyxoviridae family that causes encephalitis in young piglets and infertility in adult sows and boars. Infertility in sows naturally infected by PoRV-LPMV is characterized by an increased number of returns to oestrus, stillbirths and mummified fetuses. In this study, nine seronegative gilts were inoculated intranasally with the PAC-3 strain of PoRV-LPMV at week 6 or 10 of gestation. These animals were then killed at weeks 8 or 15 of gestation (seven gilts) or after natural parturition (two gilts). Four control gilts were mock-infected at gestation week 6 or 10 and killed between 2 and 4 weeks later. Gross lesions of focal congestion and haemorrhage were seen in the placenta and endometrium of one gilt infected at gestation week 6 and one infected at gestation week 10. PoRV-LPMV was isolated, at 2-6 weeks post-inoculation (pi), from lung, tonsils, ovary, placenta, uterus and lymph nodes of three of the gilts infected at gestation week 6 and at 2-3 weeks pi from lung, tonsil and ovary of two gilts infected at gestation week 10. Many of the fetuses of eight infected gilts were smaller than normal and had dermal ecchymoses. Dehydrated or mummified fetuses were present in six of the infected gilts but not in any control animal. PoRV-LPMV was isolated from brain, lung and liver of fetuses from two gilts infected at gestation week 6, and from two infected at gestation week 10. These results indicate that, after experimental infection, PoRV can replicate in tissues of seronegative pregnant gilts, cross the placenta, and cause fetal death and mummification.

Apoptosis in lymph nodes and changes in lymphocyte subpopulations in peripheral blood of pigs infected with porcine rubulavirus.

In a first experiment, five pigs were inoculated intranasally with porcine rubulavirus (PoRV) at 5 days of age and killed 7 days post-infection (pi). In a second experiment, four pigs were infected with the same virus at 17 days of age and killed at 9 or 15 days pi. Control piglets in each experiment received uninfected cell culture supernate. All PoRV-infected pigs developed respiratory and nervous signs, and histological lesions of non-suppurative encephalitis and interstitial pneumonia. All control pigs remained clinically normal and did not have histological lesions. Significantly increased numbers of apoptotic cells were detected by terminal deoxynucleotidyl transferase biotin-dUTP nick end labelling (TUNEL) in tonsil and lymph nodes of the pigs infected at 7 days of age and killed at 7 days pi. Significantly increased percentages of CD2(+) and CD8(+) T lymphocytes were also found in peripheral blood of these animals at this time, while the percentages of CD4(+) and MHC class II lymphocytes were significantly reduced. Significantly increased numbers of apoptotic cells were detected in lymphoid tissues of the pigs infected at 17 days of age and killed at 9 days pi. The percentages of CD2(+), CD8(+) and MHC class II lymphocytes in peripheral blood were also significantly increased at this time; the percentage of MHC class II lymphocytes remained elevated at 15 days pi. These results indicate that induction of apoptosis is an important mechanism in the pathogenesis of PoRV infection in young pigs, and that this virus induces changes in lymphocyte subpopulations in peripheral blood.

Uptake of porcine rubulavirus (LPMV) by PK-15 cells.

BACKGROUND: The porcine virus denominated La Piedad Michoacan Virus (LPMV) is a member of the family Paramyxoviridae and is the cause of a disease in pigs present only in Mexico. The disease is characterized by meningoencephalitis and respiratory distress in young pigs, epididymitis and orchitis in boars, and reproductive failure and abortion in sows. METHODS: The cytopathology, morphology, and distribution of the hemagglutination neuraminidase (HN) and nucleoprotein (NP) proteins of LPMV were investigated following inoculation into PK-15 cells. The cytopathic effect was characterized by cytoplasmic vacuolation and the formation of syncytia and cytoplasmic inclusion bodies. RESULTS: In immunofluorescence assays using a monoclonal antibody (MAb) against the HN protein at 5-60 min post-infection (early infection), a diffuse immunofluorescence was observed near the cell membrane and adjacent to the nuclear membrane. At 24 h post-infection (late infection), a dust-like immunofluorescence was observed throughout the cytoplasm. LPMV-infected cells incubated with the MAb against the NP protein showed punctate cytoplasmic fluorescence during the early stages of infection. At the late infection stage, these fluorescent particles became larger and were seen predominantly in the cytoplasm of syncytia. This pattern was also apparent by immunohistochemical labeling and immunogold electron microscopy. The latter technique revealed that HN protein was diffusely distributed throughout the cytoplasm. When using the MAb against the NP protein, nucleocapsid organization was the most prominent feature and resulted in the formation of cytoplasmic inclusion bodies visible by light and electron microscopy. Immunogold labeling of purified nucleocapsids was shown by electron microscopy. Virus particles and nucleocapsids were morphologically similar to members of the Paramyxoviridae family. CONCLUSIONS: The morphologic characteristics of the virions and the distribution patterns of the HN and NP proteins in PK-15 infected cells indicate that the mechanisms of LPMV replication are generally similar to those of the members of the Paramyxoviridae family.

Trichomonas vaginalis: chromatin and mitotic spindle during mitosis.

The mitotic phases and the changes that the chromatin and mitotic microtubules undergo during mitosis in the sexually transmitted parasite Trichomonas vaginalis are described. Parasites arrested in the gap 2 phase of the cell cycle by nutrient starvation were induced to mitosis by addition of fresh whole medium. [(3)H] Thymidine labeling of trichomonad parasites for 24 h showed that parasites have at least four synchronic duplications after mitosis induction. Fixed or live and acridine orange (AO)-stained trichomonads analyzed at different times during mitosis by epifluorescence microscopy showed that mitosis took about 45 min and is divided into five stages: prophase, metaphase, early and late anaphase, early and late telophase, and cytokinesis. The AO-stained nucleus of live trichomonads showed green (DNA) and orange (RNA) fluorescence, and the nucleic acid nature was confirmed by DNase and RNase treatment, respectively. The chromatin appeared partially condensed during interphase. At metaphase, it appeared as six condensed chromosomes, as recently reported, which decondensed at anaphase and migrated to the nuclear poles at telophase. In addition, small bundles of microtubules (as hemispindles) were detected only in metaphase with the polyclonal antibody anti-Entamoeba histolytica alpha-tubulin. This antibody showed that the hemispindle and an atractophore-like structure seem to duplicate and polarize during metaphase. In conclusion, T. vaginalis mitosis involves five mitotic phases in which the chromatin undergoes different degrees of condensation, from chromosomes to decondensed chromatin, and two hemispindles that are observed only in the metaphase stage.

Immunity to porcine rubulavirus infection in adult swine.

The immune response against the porcine rubulavirus was analyzed in experimentally infected adult pigs. High titers of virus neutralizing and hemagglutinating inhibitory antibodies were identified in infected animals. The antibody specificity was directed towards HN, M, and NP rubula virion proteins; immunodominance of HN proteins was demonstrated. Peripheral blood mononuclear cells from infected, but not from non-infected pigs proliferated in vitro in response to virus antigenic stimuli, showing a bell-shaped plot with the highest peak at 5 weeks post-infection. Virus-induced lymphoblasts expressed CD4+ CD8+ phenotype, whereas lectin-induced lymphoblasts were mainly identified as CD4+ CD8- cells. Phenotype analysis of freshly prepared PBMC revealed increased number of both monocytes (PoM1+) and total T lymphocytes (CD2+) early during infection, with reduced values of B lymphocytes at 4 weeks post-infection. Decrease in CD4+ CD8- blood cells was observed at 3 weeks post-infection, whereas both CD4- CD8+ and CD4+ CD8+ cells increased 1 and 4 weeks post-infection, respectively. This work discusses the relevance of CD4+ CD8+ T cells in the control of porcine rubulavirus infection.

Immunologic, microscopic, and molecular evidence of Encephalitozoon intestinalis (Septata intestinalis) infection in mammals other than humans.

Encephalitozoon intestinalis (Septata intestinalis) is the second most prevalent microsporidian species infecting humans, but it has not been described in other animal species. This investigation examined 10 domestic animal stool samples (8 mammalian, 2 avian) containing spores detected by anti-Encephalitozoon monoclonal antibody immunofluorescence (FA). The presence of E. intestinalis but not Encephalitozoon hellem or Encephalitozoon cuniculi was confirmed in 6 of 8 mammalian stool samples by species-specific FA and polymerase chain reaction. Clusters of spores inside epithelial cells were observed in feces of five mammals (donkey, dog, pig, cow, and goat) using "quick-hot" Gram-chromotrope stain. None of the 10 samples reacted with anti-E. hellem or anti-E. cuniculi sera, nor were they amplified with species-specific primers for E. hellem and E. cuniculi. To our knowledge, this is the first identification of E. intestinalis in animals other than humans. The data shown herein suggest the possibility that E. intestinalis infection may be zoonotic in origin.

Lesions in the reproductive tract of boars experimentally infected with porcine rubulavirus.

"Blue eye" disease of pigs in Mexico is caused by porcine rubulavirus and characterized by infertility in sows and boars, nervous signs in young pigs, and corneal opacity in pigs of all ages. The pathogenesis of reproductive tract lesions in rubulavirus-infected boars has not previously been investigated. In a first experiment, four 9-month-old boars were inoculated with porcine rubulavirus and killed 5, 15, 30 or 45 days post-inoculation (pi). In a second experiment, four similar boars were inoculated with the same virus and two animals were killed on each of days 70 and 80 pi. Swelling of the head of the epididymis developed in all inoculated boars at approximately day 15 pi. Reduced spermatozoan motility and concentration were detected in semen samples collected from one boar from day 21 pi. At post-mortem examination, nodules were seen in the head of the epididymis of the boars killed 15, 30 or 45 days pi and the right testis of the pig killed 30 days pi was atrophic. Corresponding histopathological epididymal alterations included formation of spermatic granulomas and vacuolar degeneration of ductular epithelium. These lesions were associated with mononuclear cell infiltration and interstitial fibroplasia. Degeneration of seminiferous tubules and interstitial mononuclear cell infiltration were seen in the atrophic testis of the pig killed 30 days pi. There was fibrosis of the head of the epididymis in all boars killed 70 or 80 days pi and one of these animals also had right testicular atrophy associated with degeneration of seminiferous tubules, lymphocytic infiltration and giant cell formation. Porcine rubulavirus antigen was detected by immunofluorescence labelling in the head of the epididymis of the pigs killed 15, 30 or 45 days pi and in one animal killed on day 70 pi. These results indicate that porcine rubulavirus can cause severe epididymo-orchitis and reduced semen quality in sexually mature boars.

NeuAc alpha 2,3gal-glycoconjugate expression determines cell susceptibility to the porcine rubulavirus LPMV.

Relevance of membrane sialoglycoconjugates as receptors for infection by the porcine rubulavirus has been determined in vitro by sugar and lectin competition assays and by inhibition of glycosylation. Our results show that NeuAc alpha 2,3Gal but not NeuAc alpha 2,6Gal inhibits the virus infectivity of Vero cells, and the virus was effectively blocked with the lectin Maackia amurensis, specific for NeuAc alpha 2,3Gal. Inhibition of the cellular glycosylation with tunicamycin, deoxinojirimycin as well as neuraminidase treatment diminishes the viral capacity to bind and infect this cell line. Dexamethasone, which promotes the activity of sialyl alpha 2,6 glycosyltransferase, also diminishes the cell susceptibility for infection. This is the first report confirming that NeuAc alpha-2,3Gal recognition is determinant in the pathogenesis of the porcine rubulavirus.

The porcine paramyxovirus LPM specifically recognizes sialyl (alpha 2,3) lactose-containing structures.

The porcine paramyxovirus LPM recognizes alpha, but not beta, anomers of sialic acid containing structures, specifically sialyl (alpha 2,3) lactose. The virus specificity is directed to the sialyl residue and to the C'4 axial OH and the C'6 CH2OH of the galactose present in this structure.

The structural proteins of a porcine paramyxovirus (LPMV).

The porcine paramyxovirus is a newly identified agent of a fatal disease in piglets, endemic in Mexico since 1980, where it was seen around the town of La Piedad, Michoacan, Mexico (hence LPM virus). At least six [35S]methionine-labelled proteins could be resolved by SDS-PAGE and five of them were clearly immunoprecipitated. Selective labelling of LPMV-infected cells with [3H]glucosamine revealed two bands with an Mr of about 66K and 59K, corresponding to the two viral glycoproteins, the haemagglutinin-neuraminidase protein and the fusion protein. Labelling of virus with [32P]orthophosphate disclosed one band with an Mr of 52K, corresponding to the phosphoprotein. Analysis of nucleocapsids obtained from purified virus or from a permanently infected cell line revealed one major band with an Mr of 68K, the nucleoprotein. Two other proteins were also identified, the large protein and the matrix protein, with apparent Mr of about 200K and 40K, respectively. The protein migration pattern of LPMV was compared, by SDS-PAGE, with that of Newcastle disease virus, bovine parainfluenza 3 virus and Sendai virus. Differences in the Mr of LPMV proteins and the proteins of these paramyxoviruses were observed. We propose that LPMV should be classified as a novel member of the genus Paramyxovirus.