Survival of Bacterial and Parasitic Pathogens from Zebrafish (Danio rerio) After Cryopreservation and Thawing.

Cryopreservation is a common method used to preserve the sperm of various animal species, and it is widely used with zebrafish (Danio rerio). As with other animals, there is a possibility of paternal pathogen transmission through sperm. We evaluated the ability of five common and important pathogens of zebrafish to survive cryopreservation as used with zebrafish sperm and freezing without cryopreservant. We evaluated Mycobacterium chelonae, Mycobacterium marinum, and Edwardsiella ictaluri, each originally isolated from zebrafish, eggs of Pseuodocapillaria tomentosa, and spores of Pseudoloma neurophilia. Each mycobacterial isolate showed relatively minimal reduction in survival after freezing and thawing, particularly when subjected to cryopreservation. E. ictaluri also showed survival after cryopreservation, but exhibited a several log reduction after freezing at -80°C without cryopreservant. With P. neurophilia, two separate experiments conducted 3 years apart yielded very similar results, showing some, but reduced, survival of spores by using three different viability assays: SYTOX stain, Fungi-Fluor stain, and presence of a spore vacuole. Eggs of P. tomentosa showed no survival based on larvation of eggs when subjected to either freezing method. Given that four of the five pathogens exhibited survival after cryopreservation, we recommend that sperm samples or donor male zebrafish fish be tested for pathogens when sperm are to be stored by using cryopreservation.

Effect of natural neosporosis on bull sperm quality.

Neospora is one of the protozoans that can infect the male and female's reproduction system. Despite the existence of N. caninum in the genitalia, its effect on sperm characteristics was not studied yet. Therefore, the aim of this study was to investigate the influence of natural neosporosis on the sperm parameters of bulls. Using 30 bulls with neosporosis diagnosed by modified agglutination test and enzyme-linked immunosorbent assay (ELISA) and 15 healthy bulls, some sperm parameters such as sperm concentration, viability, motility, and morphology were studied and compared. Also, the activity of super oxide dismutase (SOD), glutathione peroxidase (GPX), and malondialdehyde (MDA) level as the biomarker of lipid peroxidation was investigated. Results showed that sperm concentration, viability, and motility were significantly lower in bulls with neosporosis in the present study. There were no significant differences in activities of SOD and MDA level but GPX activity was significantly increased in infected bulls.

Susceptibility of gametes and embryos of the eastern oyster, Crassostrea virginica, to Karenia brevis and its toxins.

The bivalve mollusc, Crassostrea virginica, is frequently exposed to blooms of Karenia brevis along the west coast of Florida during periods of spawning and early larval development. A continuous 4-day exposure of gametes and 2-4 cell stage embryos of C. virginica to whole-cell and culture filtrate of K. brevis at 500 and 5000 cells mL(-1), was followed by a 4-day 'recovery' period. Larval growth, percent of normal, abnormal and dead larvae, and the presence of food in the larval gut were measured throughout the exposure period. Results suggest that negative effects mainly occur during embryogenesis and early development. Damage to feeding apparatus/gut may occur during embryonic development or exposure to toxins may act as a feeding deterrent on non-toxic algae. Following 2-h in vitro exposure of gametes, differences in oocyte and sperm cell parameters were investigated using flow cytometry. The reduced sperm viability in the whole-cell 5000 cells mL(-1) treatment suggests the involvement of extracellular brevetoxins (PbTx) and perhaps other harmful, uncharacterized compounds associated with the K. brevis cell membrane. The cumulative effects of reduced sperm viability, fertilization success, embryonic and larval survival, and the near-annual exposure to blooms of K. brevis could cause significant bottlenecks on oyster recruitment.

Relationship between testicular lesion and hormone levels in male rats infected with Trypanosoma evansi.

The aim of this study was to evaluate the relationship between testicular lesions and hormone levels in rats experimentally infected with Trypanosoma evansi. For that, the measurement of reproductive hormones, histopathology and biomarkers of cellular injury were carried out in twenty-four animals, which were divided into two groups with 12 animals each. Group A was the negative control, or uninfected, while group B was composed by animals infected with T. evansi. Both groups were divided again into two other subgroups (n=6), from which serum and testicular fragments were collected on days 5 (A1 and B1) and 15 (A2 and B2) post-infection (PI). The morphological analysis showed increased alterations of head and tail of sperm in infected rats when compared with those of the control group. A significant reduction (P<0.01) in the levels of LH, FSH, testosterone and estradiol, associated with an increase in cortisol, was observed in serum of group B when compared with negative control. Additionally, NOx, lipid peroxidation and protein oxidation were enhanced in testicles, indicating the occurrence of cellular lesion. On histopathology, it was possible to observe testicular degeneration, among other disorders in infected animals. Therefore, based on these results, it is possible to conclude that the experimental infection with T. evansi caused changes in the levels of the main hormones of male rats associated with cellular injury.

Infecciones de transmisión sexual en semen: el hombre como vector de transmisión / Sexual transmission infections in semen: men as vector transmission

En los últimos años el estudio de las infecciones de transmisión sexual ha cobrado gran importancia debido principalmente al incremento de estas en parejas heterosexuales y hombres que tienen sexo con hombres. En mujeres existe mucha información de epidemiología y patogénesis de estas infecciones, sin embargo, en hombres la información es muy escasa debido a que la mayoría no presenta sintomatología. En los últimos años se ha evidenciado un creciente interés en el estudio del semen como vía de transmisión, debido principalmente a la afinidad de algunos patógenos con los espermatozoides. Dentro de los principales microorganismos infectantes en semen se encuentran Chlamydia trachomatis, Neisseria gonorrhoeae, Mollicutes, Virus de la Inmunodeficiencia Humana tipos 1 y 2, Virus Herpes Simplex 1 y 2, Virus Papiloma Humano, Virus de la Hepatitis B y C, Citomegalovirus, Virus Epstein-Barr y Trichomonas vaginalis.

Sexually transmitted infections study has become an important issue in these days, mainly due to the increment of heterosexual and men have sex with men partners of people. In women, there is a lot information about epidemiology and pathogenesis of these infections. However, the information is very limited in men, because most infected men are asymptomatic. In recent years, there has been an increasing interest in study of semen as a transmission way, due to the affinity of some pathogens to sperm. The most prevalent microorganisms infecting semen are: Chlamydia trachomatis, Neisseria gonorrhoeae, Mollicutes, Human Immunodeficiency Virus Types 1 and 2 Herpes Simplex Virus 1 and 2, Human Papillomavirus, Hepatitis B and C virus, Cytomegalovirus, Epstein-Barr and Trichomonas vaginalis.

Sex-specific effects of a parasite evolving in a female-biased host population.

BACKGROUND: Males and females differ in many ways and might present different opportunities and challenges to their parasites. In the same way that parasites adapt to the most common host type, they may adapt to the characteristics of the host sex they encounter most often. To explore this hypothesis, we characterized host sex-specific effects of the parasite Pasteuria ramosa, a bacterium evolving in naturally, strongly, female-biased populations of its host Daphnia magna. RESULTS: We show that the parasite proliferates more successfully in female hosts than in male hosts, even though males and females are genetically identical. In addition, when exposure occurred when hosts expressed a sexual dimorphism, females were more infected. In both host sexes, the parasite causes a similar reduction in longevity and leads to some level of castration. However, only in females does parasite-induced castration result in the gigantism that increases the carrying capacity for the proliferating parasite. CONCLUSIONS: We show that mature male and female Daphnia represent different environments and reveal one parasite-induced symptom (host castration), which leads to increased carrying capacity for parasite proliferation in female but not male hosts. We propose that parasite induced host castration is a property of parasites that evolved as an adaptation to specifically exploit female hosts.

Ejaculate characteristics of rabbits infected with Trypanosoma congolense and changes caused after treatment with Diminazene aceturate (Diminaveto®) / Características eyaculatorias de conejos infectados con Trypanosoma congolense y cambios causados después del tratamiento con Diminazeno aceturato (Diminaveto®)

The effects of experimental Trypanosoma congolense infection on the ejaculate of rabbits and changes caused after treatment with Diminaveto® were investigated using 24 New Zealand White rabbits (bucks). The bucks were housed singly in standard rabbit cages and fed on specialized ration containing 10 percent Protein supplement, grains, legume, salt and fresh water ad libitum during the study. Data on ejaculate characteristics were collected from all the bucks in the first phase (i.e. before infection) and in the second phase (i.e. during infection, with 4.8x10(5) Trypanosoma congolense, intraperitoneally). Similar data were collected from 12 randomly selected bucks treated with 7.0mg/kg Diminaveto® following reconstitution during the third phase. Data collected were analysed using the Paired T- Test and Analysis of Variance. The infection led to significant (P< 0.05) reduction in spermatozoa motility, concentration and mass activity, with a significant (P< 0.05) increase in percentage of sperm cells with morphological abnormalities. Treatment with Diminaveto® led to improvement in all ejaculate parameters investigated. However, it was observed that the ejaculate did not attain the "before-infection" status following treatment with Diminaveto®. The study showed that infection with Trypanosoma congolense in rabbits caused significant reduction in ejaculate characteristics. Treatment with Diminaveto® however led to improvement in the ejaculate though at a rate slower than that at which the infection caused the reduction.

Fue estudiado en 24 conejos Nuevo zelandeses blancos, machos, los efectos de la infección experimental de Trypanosoma congolense sobre la eyaculación y los cambios producidos después del tratamiento con Diminaveto® . Los machos fueron colocados individualmente en jaulas de conejos estándar. Durante el estudio fueron alimentados con ración especializada, con 10 por ciento de suplemento de proteínas, granos, legumbres, sal y agua fresca ad libitum. Los datos sobre las características de la eyaculación se obtuvieron de todos los machos en la primera fase (es decir, antes de la infección) y en la segunda fase (es decir, durante la infección, con 4,8x10(5) Trypanosoma congolense, por vía intraperitoneal). Datos similares se obtuvieron de 12 machos al seleccionados al azar, tratados con Diminaveto® 7,0mg/kg después de la reconstitución durante la tercera fase. Los datos fueron analizados mediante t de student y análisis de varianza. La infección fue significativa (P <0,05) habiendo reducción de la motilidad de los espermatozoides, la concentración y actividad de masas, con un efecto significativo (P <0,05) aumento en el porcentaje de espermatozoides con anomalías morfológicas. El tratamiento con Diminaveto® condujo a una mejoría en todos los parámetros investigados del eyaculado. Sin embargo, se observó que el eyaculado no alcanzó el "antes de la infección" tras el tratamiento con Diminaveto®. Además, el estudio mostró que la infección con Trypanosoma congolense en conejos causó una reducción significativa en las características del eyaculado. Sin embargo, el tratamiento con Diminaveto ® condujo a una mejoría en la eyaculación aunque a un ritmo más lento que en la infección causada por la reducción.

DNA types of aspermic Fasciola species in Japan.

In order to reveal DNA types of aspermic Fasciola forms in Japan, Fasciola specimens obtained from eight prefectures that had not been previously reported were analyzed for DNA of ribosomal internal transcribed spacer 1 (ITS1) and mitochondrial NADH dehydrogenase 1 (ND1) gene. Five combinations in DNA types of both ITS1 and ND1 were revealed from the results of this study and previous studies. The DNA type Fsp2, which is identical to that of F. gigantica in both ITS1 and ND1, was the most predominant in Japan, followed by Fsp1, which is the same DNA type as that of F. hepatica. Fasciola forms with Fsp1 mainly occurred in the northern region of Japan and those with Fsp2 were mainly in the western region. The founder effect related to migration of definitive host and susceptibility of intermediate host snail might play an important role in both geographical distribution and frequency of DNA types in Japanese Fasciola specimens.

Tritrichomonas fetus extracellular products decrease progressive motility of bull sperm.

Tritrichomonas fetus causes infertility and abortion in cattle; however, there is scarce information regarding the susceptibility of bovine sperm to this parasite. The objective of this study was to analyze in vitro the interaction between T. fetus and bovine sperm and to evaluate the effect of extracellular products secreted by the parasite on these reproductive cells. Sperm from five fertile bulls (Bos taurus taurus, Holstein-Friesian), selected through a Percoll gradient, adhered to T. fetus after 30min of interaction, resulting in agglutination between the two kinds of cells. Based on reverse transcription-polymerase chain reaction (RT-PCR), T. fetus continuously expressed its gene for cysteine peptidase in the presence or absence of sperm. Computer-assisted semen analysis (CASA) revealed that, after 1h incubation of sperm in T. fetus culture extract, the extracellular products secreted by the parasite decreased sperm progressive motility (P<0.05). Although T. fetus extracellular products did not lead to loss of sperm viability (P<0.05) based on the Annexin-V/propidium iodide assay, the percentage of Annexin-V fluorescein isothiocyanate-positive and propidium iodide-positive cells increased (P<0.05) during incubation of sperm in T. fetus culture extract, consistent with cellular damage. In conclusion, extracellular products secreted by T. fetus were cytotoxic to bovine sperm, as they decreased sperm progressive motility; perhaps this contributes to the pathogenesis of T. fetus-induced infertility.

Semen variables of sheep (Ovis aries) experimentally infected with Toxoplasma gondii.

The influence of Toxoplasma gondii on semen variables and sperm morphology of sheep was evaluated in eight reproductive males distributed into three experimental groups: GI, three sheep inoculated with 2.0x10(5) of P strain oocytes; GII, three sheep infected with 1.0x10(6) of RH strain tachyzoites and; GIII two control sheep. Clinical (rectal temperature, cardiac and respiratory frequencies), parasite and serology exams (IIF) were realized. Sperm variables (volume, motility, vigor and concentration) and semen morphology for each sheep were also evaluated. Thus, semen and blood collections were assessed on post-inoculation days (PIDs)-1,3,5,7,11,14 and weekly thereafter up to PID 70. Clinical alterations were observed (hypothermia and anorexia) in infected sheep from groups GI and GII. Parasitic outbreaks were detected in five sheep. All the infected sheep produced antibodies against T. gondii from PID 5 onwards, reaching a peak of 4096 and 8192 for group GI and GII sheep, respectively. Differences (P<0.05) were observed regarding the ejaculate volume between the inoculated groups (oocytes and tachyzoites) and control. Even though experimental toxoplasmic infection resulted in clinical symptomology in the inoculated sheep, the minimal alterations in sperm pathologies could not be directly attributed to T. gondii.

Counteraction during movement of spermatozoa by Trichomonas vaginalis observed by visual image analysis: a possible cause of female infertility.

OBJECTIVE: To use new medical imaging technology to predict the motility pattern of Trichomonas vaginalis within the vagina. DESIGN: Use of IPAN Tracker IP97, a noniterative, competitive linking algorithm. SETTING: University laboratory. PATIENT(S): None. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Visualization of movement pattern of protozoon within the vagina. RESULT(S): The motility pattern of T vaginalis within the vagina is a vertical zigzag configuration that results in a circular whirling. During movement of spermatozoa, its counteraction by T vaginalis could be visualized. CONCLUSION(S): T vaginalis has flagella and is mobile within the vagina; its whirling movement can disrupt normal sperm movement and potentially result in difficulty in fertilization.

Trichomonas adhere and phagocytose sperm cells: adhesion seems to be a prominent stage during interaction.

Tritrichomonas foetus and Trichomonas vaginalis are extracellular parasites of the urogenital tract of cattle and humans, respectively. They cause infertility and abortion, but there is no documented information on the susceptibility of bovine sperm cells to this cattle parasite. The aim of this present work was to study the effects provoked by T. foetus and T. vaginalis when in interaction with bovine and human sperm cells. The bovine and human spermatozoa were obtained from uninfected bulls and men, respectively, and were exposed to living trichomonads over different periods of time. Light microscopy, video microscopy, scanning, and transmission electron microscopy first revealed a tropism, then a close proximity followed by a tight adhesion between these two different cells. A decrease in the spermatozoa motility was observed as well intense semen agglutination. The adhesion between trichomonads to the sperm cell occurred either by the flagella or sperm head. Motile parasites were observed during the next 12 h, whereas sperm cells in contact with the parasites rapidly became immotile. The parasites were able to maintain the sperm cells attached to their cell surface, followed by phagocytosis. This process began with a tight membrane-membrane adhesion and the incorporation of the sperm cell within an intracellular vacuole. Afterwards, the sperm cell was gradually digested in lysosomes. Many trichomonads were injured and/or died on making contact with the spermatozoa possibly due to necrosis. Results from this study demonstrated that both T. foetus and T. vaginalis interact with sperm cells provoking damage and death of these reproductive cells. Differences in the behavior of both trichomonads were evident, showing that T. vaginalis was much more virulent than T. foetus. The possible role of trichomonads in reproductive failure is discussed.

Sexually mediated shedding of Myxobolus fallax spores during spermiation of Litoria fallax (anura).

Myxobolus fallax (Myxosporea) infects the testes of the dwarf green tree frog Litoria fallax without apparently affecting the host's health, behavior, or testicular sperm numbers or quality. We investigated the shedding M. fallax spores and the relationship between M. fallax infection and fertility in L. fallax. During natural spawning, comparisons were made between the prevalence and intensity of M. fallax infection, spore shedding, and fertilization rates. During the hormonal induction of spermiation, comparisons were made between the prevalence and intensity of M. fallax infection, and the number of sperm and spores shed. During natural spawning, the prevalence or intensity of infection or spore shedding did not affect fertility. Spermiation and spore shedding was induced in 10 males by the administration of human chorionic gonadotrophin, with 10 controls. Histology showed that all 10 males were infected by M. fallax. The controls shed no sperm or spores. Nine infected males shed sperm 3 h post-administration (PA), and 3 of these also shed spores. Only the 3 males that shed sperm and spores at 3 h PA also shed sperm at 6 h PA, at which time they also shed spores. In total, the 3 males that shed spores gave higher sperm numbers than the 6 males that did not shed spores. Overall, these results show that the shedding of M. fallax spores is sexually mediated through reproductive hormones without affecting fertility.

The effects of trypanosomosis on sperm morphology in Zebu x Friesian crossbred bulls.

Detailed studies of sperm morphological abnormalities were carried out on 12 Zebu x Friesian crossbred bulls used in a study of the effects of trypanosomosis. Four bulls were infected with T. vivax, another four with T. congolense, while four served as controls. The infected bulls developed chronic trypanosomosis. All the bulls initially had very low sperm morphological abnormalities that were within acceptable limits for fertile animals. After infection there was a rapid and progressive increase in all sperm abnormalities. Spermatozoa of infected bulls were highly deformed with multiple morphological defects. Mean percentage pre-infection baseline values prior to infection for acrosomal, sperm-head, detached heads, proximal cytoplasmic droplets, distal cytoplasmic droplets, sperm-tail, midpiece and total sperm morphological defects ranged between 0.1 +/- 0.1 for acrosomal and 8.3 +/- 3.2 for total morphological abnormalities in the semen of the bulls. All the infected bulls developed sperm morphological abnormalities of more than a mean of 40.0% from the 4th week after infection until the end of the investigation and were considered unfit for breeding. At 7 weeks post-infection (PI) until the end of the study (12 weeks PI), the controls had a mean of less than 5% sperm morphological defects, while the infected bulls had 100%. Mean percentage values of sperm morphological defects throughout the duration of the investigation for control bulls were low and within the normal range for fertile bulls. These values differed significantly (p<0.001) from the elevated values of the infected bulls. The results show that trypanosomosis due to T. vivax or T. congolense infection can render Zebu x Friesian crossbred bulls unfit for breeding within a very short time. The resultant infertility could be of economic importance in trypanosomosis-endemic sub-Saharan Africa where Zebu x Friesian crossbred bulls are kept.

Ultrastructure of spermiogenesis and the spermatozoon of Nomimoscolex sp. (Cestoda, Proteocephalidea) intestinal parasite of Clarotes laticeps (Fish, Teleost) in Senegal.

Spermiogenesis in Nomimoscolex sp. begins with the formation of a differentiation zone delimited at the front by arched membranes. It is bordered by cortical microtubules and contains two centrioles surmounted each by striated roots. The latter are linked together at their bases by a thin layer of electron-dense material. The two centrioles give each a flagellum that rotates and fuses with the median cytoplasmic extension. The nucleus elongates, becomes filiform and migrates between the axonemes in the cytoplasmic extension. During the nuclear migration, crested-like bodies form. After the migration of the nucleus, the old spermatid separates from the residual cytoplasm by strangulation of the ring of arched membranes. The mature spermatozoon of Nomimoscolex sp. lacks mitochondria, is filiform and tapered at both its extremities. Its anterior end exhibits an apical cone of electron-dense material and three crested-like bodies of unequal length coiled in a spiral on its periphery. The axoneme is of the 9 + 1' pattern. The cortical microtubules are parallel to the spermatozoon axis. The nucleus is a fibrous cord of partly condensed chromatin. The cytoplasm is slightly dense but contains many electron-dense granules in regions II and III of the spermatozoon. Crested-like bodies and striated roots linked together by an electron-dense material have never been reported during the spermiogenesis in a proteocephalidean. In addition we describe for the first time the existence of three crested-like bodies in the Proteocephalidea.

Detection of Trichinella pseudospiralis antigen in the enteral phase in experimentally infected mice.

The distribution of T. pseudospiralis antigen in tissue sections of the intestine of infected mice was studied at different intervals (10, 21, 35 hours and 10 days post-infection (P.I.). Immunocytolocalization of antigen in the intestine was done by peroxidase antiperoxidase staining technique. Positive staining reaction of the enterocytes was noted in all durations examined. The mononuclear cells of the lamina propria were positively stained at 10 hrs P.I. while patchy staining of muscularis mucosa was observed at 10 days P.I. The present study revealed intense staining reaction in the stichosome and the cuticle of 10 hrs-old larvae L1, the intensity of the reaction decreased throughout the worm development, until it almost disappeared at 10 days P.I. The gut lining and its occupying substance were positive throughout the enteral phase. Intense staining reaction of the hypodermal cells was observed in L2, L3 and adult stage worm. The genital primordium was weakly stained in the larval stages while the male and female reproductive systems were moderately stained at the adult stage.