Stochastic processes govern bacterial communities from the blood of pikas and from their arthropod vectors.

Vector-borne microbes influence pathogen transmission and blood microbiomes, thereby affecting the emergence of infectious diseases. Thus, understanding the relationship between host and vector microbiomes is of importance. In this study, we investigated the bacterial community composition, diversity and assembly of the flea (Rhadinopsylla dahurica vicina), torsalo (Hypoderma curzonial), and the blood and gut of their shared pika host, Ochotona curzoniae. Bartonella, Sphingomonas and Bradyrhizobium were enriched in blood, while Wolbachia and Fusobacterium were more abundant in fleas and torsaloes. Most of potential pathogenic microbes (belonging to Fusobacterium, Rickettsia, Kingella, Porphyromonas, Bartonella and Mycoplasma) were present in the blood of pikas and their vectors. Blood communities were more similar to those from fleas than other sample types and were independent of host factors or geographical sites. Notably, blood microbes originate mainly from fleas rather than gut or torsaloes. Interestingly, the community assembly of blood, fleas or torsaloes was primarily governed by stochastic processes, while the gut microbiome was determined by deterministic processes. Ecological drift plays a dominant role in the assembly of blood and flea microbiomes. These results reflect the difficulty for predicting and regulating the microbial ecology of fleas for the prevention of potential microbiome-associated diseases.

Inhibition of inducible nitric oxide synthase expression and nitric oxide production in plateau pika (Ochotona curzoniae) at high altitude on Qinghai-Tibet Plateau.

Nitric oxide (NO), a potent vasodilator, plays an important role in preventing hypoxia induced pulmonary hypertension. Endogenous NO is synthesized by nitric oxide synthases (NOSs) from l-arginine. In mammals, three different NOSs have been identified, including neuronal NOS (nNOS), endothelial NOS (eNOS) and inducible NOS (iNOS). Plateau pika (Ochotona curzoniae) is a typical hypoxia tolerant mammal that lives at 3000-5000 m above sea level on the Qinghai-Tibet Plateau. The aim of this study was to investigate whether NOS expression and NO production are regulated by chronic hypoxia in plateau pika. Quantitative real-time PCR and western blot analyses were conducted to quantify relative abundances of iNOS and eNOS transcripts and proteins in the lung tissues of plateau pikas at different altitudes (4550, 3950 and 3200 m). Plasma NO metabolites, nitrite/nitrate (NO(x)⁻) levels were also examined by Ion chromatography to determine the correlation between NO production and altitude level. The results revealed that iNOS transcript levels were significantly lower in animals at high altitudes (decreased by 53% and 57% at altitude of 3950 and 4550 m compared with that at 3200 m). Similar trends in iNOS protein abundances were observed (26% and 41% at 3950 and 4550 m comparing with at 3200 m). There were no significant differences in eNOS mRNA and protein levels in the pika lungs among different altitudes. The plasma NO(x)⁻ levels of the plateau pikas at high altitudes significantly decreased (1.65±0.19 µg/mL at 3200 m to 0.44±0.03 µg/mL at 3950 m and 0.24±0.01 µg/mL at 4550 m). This is the first evidence describing the effects of chronic hypoxia on NOS expression and NO levels in the plateau pika in high altitude adaptation. We conclude that iNOS expression and NO production are suppressed at high altitudes, and the lower NO concentration at high altitudes may serve crucial roles for helping the plateau pika to survive at hypoxic environment.

Rickettsial infections of dogs, horses and ticks in Juiz de Fora, southeastern Brazil, and isolation of Rickettsia rickettsii from Rhipicephalus sanguineus ticks.

The present study was performed in an area endemic for Brazilian spotted fever (BSF) in Juiz de Fora, state of Minas Gerais, Brazil, during the years 2007 and 2008, when fatal cases of BSF (caused by Rickettsia rickettsii) were reported. Adult ticks (Acari: Ixodidae) identified as Rhipicephalus sanguineus (Latreille) and Amblyomma cajennense (Fabricius) were collected from dogs and horses, respectively, and tested by polymerase chain reaction (PCR). Overall, 13.1% of the Rh. sanguineus ticks and none of the A. cajennense were found to be infected with R. rickettsii. Two isolates of R. rickettsii were successfully established in Vero cell culture from two Rh. sanguineus ticks. An indirect immunofluorescence assay (IFA) using R. rickettsii antigens detected blood serological reaction to R. rickettsii in 67.9% (53/78) of dogs and 41.0% (16/39) of horses living in the study area. Larval offspring from two Rh. sanguineus engorged females, naturally infected by R. rickettsii, were reared to adult stage in the laboratory. All active stages (larvae, nymphs, adults) remained 100% infected by R. rickettsii, which was efficiently transmitted to naïve rabbits. Overall, the results of the present study indicate a potential risk for transmission of R. rickettsii to humans by Rh. sanguineus, an occurrence yet to be documented in Brazil.

The ELISA for the examination of hare sera for anti-Brucella antibodies.

Hare brucellosis is caused primarily by Brucella suis biovar 2. Hares along with wild boars are the natural reservoir of this microorganism. In view of restriction of applicability of traditional serological methods the work aimed to develop the ELISA to examine hare sera for the presence of anti-Brucella antibodies. Lipopolysaccharide (LPS) antigen obtained from the strain S19 of Brucella abortus and the conjugate of antibodies against rabbit immunoglobulin with horseradish peroxidase were used in the test. Hares' sera positive and negative in the CFT were used as controls of the ELISA. The sera collected from 9 hares suspected to be infected with Brucella organisms, positive in CFT (in this number 7 hares revealed clinical symptoms or anathomopathological lesions characteristic of brucellosis), 6 sera from hares showing no symptoms of the disease, negative in CFT and 520 sera from hares monitored for brucellosis were tested. All serum samples from hares suspected for Brucella infection were positive in ELISA and 2 of them were negative in RBPT. Additionally among the samples from hares monitored 12 sera were positive in ELISA and CFT, whereas 9 sera from 12 ones were also positive in the RBPT. The obtained results indicated that the ELISA developed in our laboratory proved to be equivalent in specificity to CFT. In addition, ELISA proved to be more sensitive than RBPT for the diagnosis of Brucella infection in hares.

The binding potential of commercial antibody conjugates with sera of various small terrestrial mammals.

Infectious diseases of wild animals are of increasing importance, both from an economic viewpoint and because several of these diseases are pathogenic to man. However, serosurveys to determine the circulation of infectious organisms in wildlife are complicated by the fact that antibodies to species-specific immunoglobulins are not available for use in serological assays such as enzyme-linked immunosorbent assays (ELISAs) or immunofluorescence assays. To determine the binding potential of four commercially available antibody conjugates with the sera of wild animals, sera from 27 species of small terrestrial mammals were allowed to react with alkaline phosphatase-labelled protein A, anti-rabbit IgG, anti-mouse IgG and anti-human IgG by by the use of an ELISA. It was found that sera from some species of the order Lagomorpha bound optimally to anti-rabbit IgG, while anti-mouse IgG could be used for most species of Rodentia. For all Carnivora, Insectivora, Macroscelidea, Hyracoidea and other Rodentia, staphylococcal protein A demonstrated optimal binding. None of the sera that was tested bound to anti-human IgG. These results demonstrate that commercial conjugates can be used in serological assays in which wild animal sera are used, and should be useful for future serosurveys to determine the circulation of infectious agents in small terrestrial mammals.

Genetic polymorphism of transferrin (TF) and the haemoglobin alpha chain (HBA) in the brown hare (Lepus europaeus).

Genetic polymorphism in transferrin (TF) and in the haemoglobin alpha chain (HBA) was detected in the brown hare (Lepus europaeus) from Austria and Czechoslovakia by means of horizontal agarose and starch gel electrophoresis, respectively. Genetic analyses of complete families suggest that the TF and the HBA systems are each controlled by one autosomal gene locus with two codominant alleles. The distribution of both polymorphisms among some free-ranging Austrian brown hare populations was examined and the observed genotypes were in good agreement with the Hardy-Weinberg expectations. A comparison between the brown hare and the rabbit revealed no bands in common. The HB beta-chain was monomorphic in all specimens investigated.

[Selected biogenic elements in the blood of hares (Lepus europaeus Pall.)]. / Vybrané biogenní prvky v krevní plazme zajecí zvere (Lepus europaeus Pall.)

Blood samples were collected through aortic punctures in hares coming from different agricultural regions. Base biogenic elements (Ca, P, Mg, Na, K, chlorides) were determined in blood plasma not showing haemolysis. The values obtained in hares born and kept in captivity are presented for the purposes of comparison. Changes in element levels are described for different pollution load of ecosystems, from physiological aspects for more advanced stages of gravidity, for a higher lactation number and for young growth. The problems of qualitative fasting of hares can also be documented by mineral contents in blood plasma. The levels of some elements were evaluated for a group of adult male and female hares, and the youngs regardless of their sex until the disappearance of Stroh's outgrowth. The results were processed statistically and are summarized in tables (Tabs. 1-12) and they are compared with available literature.

Blood chemistry and endoparasites of the mountain hare (Lepus timidus L.) in high and low density populations.

In order to study the effect of high population density on the condition, blood characteristics and helminth parasitism of mountain hares (Lepus timidus), 12 specimens were shot in December 1982 and 12 more in February 1983 on the west coast of central Finland (group 1, dense population). In addition 14 hares were shot in December 1982 about 100 km from group 1 (group 2, dense population). Group 3 consists of 15 hares from stable, rather low density populations shot in southern Finland during three previous winters. The hares in group 1 were the lightest, had the least fat and were the most seriously infected with Protostrongylus pulmonalis and Trichostrongylus retortaeformis, while those in group 2 were the heaviest and had the highest Ca, Mg, alkaline phosphatase and creatinine values. The group 3 hares had the most fat. The group 1 animals shot in February 1983 had higher Ca, Mg, triglyceride and cholesterol values than those shot in December 1982. It seems that high population density combined with a lack of suitable food leads to poor condition and high endoparasite abundances. The differences in Ca and Mg are probably due to diet. The higher creatinine values in group 2 and in the hares with little or no T. retortaeformis infection may be due to the greater muscle mass.

Gonadotrophins and sex steroids during pregnancy and natural superfoetation in captive brown hares (Lepus europaeus).

In brown hares, which are induced ovulators, sexual behaviour occurs episodically at the beginning of pregnancy. From Day 34 (length of pregnancy is 41 days), the frequency of sexual chases followed by mating, ovulation and fertilization increased and 59% of pregnant females presented a natural superfoetation. The pattern of circulating luteinizing hormone (LH), follicle-stimulating hormone (FSH), oestradiol and progesterone was studied in 13 pregnant females left permanently with a male, and in 10 females isolated from males around Day 20 of pregnancy. In the 2 groups FSH concentrations were high at the beginning and end of pregnancy. All females presented a peak value of FSH in the last 4 days of pregnancy, regardless of mating stimuli. This peak value was higher for females left permanently with a male than for isolated ones. Oestradiol concentrations fluctuated between 20 and 100 pg/ml, without any clear correlation with sexual behaviour, stage of pregnancy or profiles of other hormones. Prepartum matings occurred when progesterone values were still greater than 50 ng/ml; they were followed by a transient rise in LH and by a periovulatory progesterone secretion, with values above 100 ng/ml in the morning after mating. Such modifications of LH and progesterone were not detected before Day 34, suggesting that mating stimuli are not able to induce an LH surge at the beginning of pregnancy. After Day 34, mating can induce an LH surge, ovulation and superfoetation.

Effect of ovariectomy at two periods of the year on LH and FSH basal concentrations and pituitary response to LHRH in the brown hare (Lepus europaeus).

In the brown hare, fertile mating takes place from the beginning of December to September. Seasonal variations of basal concentrations of LH and FSH, and pituitary response to a monthly i.v. injection of LHRH were studied in intact control females and in females ovariectomized during the seasonal anoestrus (OVX1) or during the breeding season (OVX2). In intact females, both basal and LHRH-stimulated LH levels showed an annual variation, with minimal values during anoestrus. During the breeding season, the LH response to LHRH exhibited a biphasic pattern. In contrast, there was no clear seasonal variation in basal and LHRH-stimulated FSH concentrations. After ovariectomy during anoestrus, basal LH remained low for 2 months and began to increase in December. After ovariectomy during the breeding season, LH basal concentrations increased within a few days after the operation. Thereafter, LH values remained high in both groups of females until September, and decreased significantly as in intact females. The pattern of LH release after LHRH remained monophasic in the two groups of ovariectomized females. In OVX1 females, the LH response increased as early as October, was maximum from December to April and decreased progressively until October. IN OVX2 females, the LH response decreased regularly after ovariectomy to a minimum in October. In the 2 groups of ovariectomized females, basal FSH concentrations and pituitary response to LHRH rose rapidly after ovariectomy and did not vary significantly thereafter. These results showed a direct central effect of season on the regulation of basal concentrations of LH, modulated by a negative feed-back of ovarian secretions during the breeding season. In intact hares, the enhanced LH response after LHRH during the breeding season was related to an acute positive effect of ovarian secretions. The regulation of FSH was less dependent on season and remained under a negative control of the ovary throughout the year.

Effect of diet on condition indices in black-tailed jackrabbits.

Changes in blood, urine and physical condition indices in 23 adult male black-tailed jackrabbits (Lepus californicus) with ad libitum feeding and 25% feed restriction were measured over a 2 wk period from 30 May to 12 June 1988. Feed restricted jackrabbits had (1) lower post-trial body weights and kidney fat indices, (2) higher femur marrow fat, serum bilirubin and cortisol concentrations, and adrenal cortex width, and (3) depressed immune function. No single index alone could best measure the nutritional status of these jackrabbits.

Pattern of serum concentrations of prolactin and progesterone during pregnancy and lactation in the brown hare (Lepus europaeus).

A heterologous radioimmunoassay system developed for the rabbit has been shown to measure prolactin in the hare. Concentrations of prolactin showed a significant (P less than 0.01) increase in the last 3 days of pregnancy (87.7 +/- 11.7 compared with 9.8 +/- 1.4 (S.E.M.) micrograms/l; n = 10 and 9 respectively) in pregnant females isolated from males, as well as in pregnant females kept with males and mating prepartum. This rise of prolactin at the end of pregnancy was not due to mating stimuli and occurred at a time when progesterone levels were still high (159 nmol/l). The injection of a slow-release preparation of bromocriptine (5 mg s.c.), which reduces prolactin secretion at the end of pregnancy, did not impair parturition. During lactation, prolactin levels increased significantly (61.2 +/- 19.8 compared with 5.3 +/- 0.1 micrograms/l; P less than 0.01) only after suckling stimuli.

Seasonal variation in the daily pattern of plasma melatonin in a wild mammal: the mountain hare (Lepus timidus).

Seasonal differences in daily patterns of plasma melatonin concentration were investigated in both free-living and captive mountain hares, in relation to reproductive activity. There was a marked increase in plasma melatonin concentrations at night at all times of the year. The period of elevation of plasma melatonin above the daytime values was longer in winter than in summer, correlating with the longer duration of darkness. The magnitude of the nighttime rise in melatonin concentrations did not differ significantly between seasons. There was no change in the plasma melatonin profile under similar photoperiods before the summer solstice when hares are sexually active or after the summer solstice when gonadal regression occurs, indicating that melatonin is not directly pro- or antigonadal. These field and laboratory observations support the view that the daily rhythm of melatonin secretion plays a role in the transduction of photoperiodic information in the mountain hare. Furthermore, they favor the hypotheses that the circadian melatonin rhythm transduces photoperiod information by means of the duration of nocturnal melatonin secretion or by the coincidence of elevated melatonin with a particular sensitive period, rather than by the amplitude of the nocturnal rise.

[Contribution to the definition of reference values in the hare: hematology -- blood biochemistry (author's transl)]. / Contribution à la définition de valeurs de référence chez le lièvre: hématologie -- biochimie sanguine.

The following hematological and biochemical reference values were determined in 150 hares: hematocrit, blood count and leucocytic formula, Na, K, Cl, P, Ca, Mg, Cu, Zn, glucose, urea, creatinine, uric acid, cholesterol, bilirubin, triglycerides, proteins and fractions, PAL, PAC, TGO, TGP, amylase, GGT, LDH. Once the effects of sex and age have been established, the references defined here will be used as a base for interpreting disturbances linked either to pathology or nutrition.

Plasma progesterone levels during pregnancy and pseudo-pregnancy in the hare (Lepus europaeus syriacus).

A triphasic pattern of progesterone secretion was observed in female hares sampled throughout pregnancy and pseudopregnancy. After injection of hCG and artificial insemination (Day 1), progesterone values rose to a peak of 41.4 ng/ml about Day 14, remained at this level, then declined around Day 20 before increasing sharply to maximum levels of 67.7 ng/ml after midpregnancy (Day 28). Levels remained high for several days, then declined until Day 38, increased again until Day 41, before decreasing towards parturition. Progesterone levels were still high (37.5 ng/ml) 24h before parturition. The progesterone pattern during pseudopregnancy closely resembled that observed during the first half of pregnancy: levels rose from Day 2 to a peak at Days 11--18, then declined sharply to baseline levels around Day 22. It is suggested that the control of progesterone secretion might be transferred from the pituitary to the placenta at the beginning of the second half of pregnancy.