老虎感染猫传染性鼻—结膜炎病毒的研究

用猫肾传代细胞从发病虎的病料中分到了一株杯状病毒粒子样病毒。该病毒大小为３５～３９ｎｍ、无囊膜、在胞浆中病毒前体呈晶格状排列,抗乙醚、不能被５－ＩＵＤＲ所抑制,能被来源于标准疫苗的猫传染性鼻－结膜炎病毒（或杯状病毒Ｆｅｌｉｎｅ ｃａｌｉｃｉｖｉｒｕｓ,ＦＣＶ）抗血清所中和,人工感染猫出现典型的ＦＣＶ感染症状。ＲＴ－ＰＣＲ能扩增出与设计值相符的电泳带,ＰＣＲ产物直接测序结果与Ｇｅｎｅｂａｎｋ发表的Ｆ     

猫泛白细胞减少症病毒的分离与鉴定

本文调查了猫科动物一种以倦怠、厌食、呕吐、下痢、血便、体温升高和白细胞数减少为特征的烈性传染病的发病规律和流行情况。利用猫肾细胞培养,从自然病例中分离出我国第一株猫泛白细胞减少症病毒——FNF8毒株。从而证实了我国有猫泛白细胞减少症的存在。     

Cytotoxic effects induced by interferon-ω gene lipofection through ROS generation and mitochondrial membrane potential disruption in feline mammary carcinoma cells

Progress in comparative oncology promises advances in clinical cancer treatments for both companion animals and humans. In this context, feline mammary carcinoma (FMC) cells have been proposed as a suitable model to study human breast cancer. Based on our previous data about the advantages of using type I interferon gene therapy over the respective recombinant DNA derived protein, the present work explored the effects of feline interferon-ω gene (fIFNω) transfer on FMC cells. Three different cell variants derived from a single spontaneous highly aggressive FMC tumor were successfully established and characterized. Lipofection of the fIFNω gene displayed a significant cytotoxic effect on the three cell variants. The extent of the response was proportional to ROS generation, mitochondrial membrane potential disruption and calcium uptake. Moreover, a lower sensitivity to the treatment correlated with a higher malignant phenotype. Our results suggest that fIFNω lipofection could offer an alternative approach in veterinary oncology with equal or superior outcome and with less adverse effects than recombinant fIFNω therapy.     

A serological survey of common feline pathogens in free-living European wildcats (<Emphasis Type="Italic">Felis silvestris</Emphasis>) in central Spain

Twenty-five serum samples of 22 free-living European wildcats (Felis silvestris) captured from 1991 to 1993 in central Spain were tested for evidence of exposure to seven feline pathogens. All the wildcats but one (95.4%) presented evidence of contact with at least one of the agents (mean = 2.2). Contact with feline leukemia virus (FeLV) was detected in 81% of the wildcats (antibodies, 77%; antigen p27, 15%). Antibodies to feline calicivirus (FCV, 80%), feline herpesvirus (FHV, 20%), feline parvovirus (FPV, 18%), and Chlamydophila sp. (27%) were also detected. Analyses were negative for feline immunodeficiency virus and feline coronavirus. The probability of having antibodies to FPV was inversely related with the concentration of serum cholesterol and with a morphometric index of body condition. Similarity in the composition of antibodies against disease agents (number and identity of detected and undetected antibodies) was significantly higher in pairs of female wildcats than in pairs of males or heterosexual pairs, suggesting that females had a more homogeneous exposure to pathogens. Seroprevalence for FHV was higher in males than in females. Antibodies to FHV and Chlamydophila sp. were more frequent in winter than in other seasons. In addition, the mean similarity of the pathogen community between pairs of serum samples was higher if both wildcats were caught during the same season than if they were not. Mean similarity was lowest when serum samples obtained in winter were compared with those from spring or summer. The results suggest that some agents probably had a reservoir in domestic cats and may cause some undetected morbidity/mortality in the studied wildcat population, whereas others, such as FeLV and FCV, may be enzootic.     

从云豹分离的一株猫泛白细胞减少症病毒的特性

从病死的云豹体内分离到1株猫泛白细胞减少症病毒,进行了鉴定,研究了它的血凝特性和对猫与水貂的致病力,分析了它与猫泛白细胞减少症病毒参考毒株(FNF-8)抗原的相关性。 用中国兽药监察所提供的猫肾传代细胞(FK)进行病毒的分离鉴定。同步接种和异步接种测得病毒的TCID50均为4.8/ml。病毒经乙醚、酸(pH3.0)、热(56℃1小时)     

The Relationship Between Coat Color and Aggressive Behaviors in the Domestic Cat

The authors explored a possible relationship between coat color and aggressive behaviors in the domestic cat. This study used an Internet-based survey to collect information on coat color, affiliative behaviors toward cats/humans, agonistic behaviors toward cats/humans, other “problem” behaviors, and cat and guardian demographic data. A total of 1,432 cat guardians completed the online survey; after exclusions based on study protocol, data analysis included 1,274 completed surveys. Guardians reported sex-linked orange female (tortoiseshells, calicos, and “torbies”), black-and-white, and gray-and-white cats to be more frequently aggressive toward humans in 3 settings: during everyday interactions, during handling, and during veterinary visits. Kruskal-Wallis 1-way analysis of variance was used to compare possible differences between the 2 sexes and among different coat colors. Analyses of aggression due to handling, as well as aggression displayed during veterinarian visits, showed little difference among coat colors in these settings.     

表达猫瘟热病毒VP2蛋白重组腺病毒的构建及其免疫原性研究

为构建能表达FPV VP2蛋白的重组犬2型腺病毒(CAV-2)载体.首先用PCR方法从FPV GT-2株细胞培养物中扩增出了VP2蛋白基因,将其克隆到真核表达质粒pVAX1中构建了含有FPV vp2基因的表达盒(CMV-VP2-PolyA),将该表达盒酶切后定向克隆到含有CAV-2 E3区的穿梭质粒pVAX△E3中,构建出pVAX△E3VP2.用Sal I+Nru I双酶切pVAX△E3VP2,回收含有目的基因表达盒部分,将其定向克隆入含有CAV-2全基因组的骨架质粒pPoly2-CAV-2中,构建了重组质粒pCAV-2-FPV-VP2.Cla I+Asc I酶切pCAV-2-FPV-VP2释放出重组基因组,以此转染MDCK细胞,获得了重组病毒CAV-2-VP2.该重组病毒能使MDCK细胞产生腺病毒样细胞病变.Western blot检测证实,该重组病毒能表达具有免疫学活性的VP2蛋白.该重组病毒可以有效地诱导免疫猫产生抗FPV和CAV-2抗体.本实验表明该重组病毒有可能成为一个FPV的疫苗株.     

表达猫瘟热病毒VP2蛋白重组腺病毒的构建及其免疫原性研究

为构建能表达FPV VP2蛋白的重组犬2型腺病毒(CAV-2)载体。首先用PCR方法从FPV GT-2株细胞培 养物中扩增出了VP2蛋白基因,将其克隆到真核表达质粒pVAX1中构建了含有FPV vp2基因的表达盒(CMV- VP2-PolyA),将该表达盒酶切后定向克隆到含有CAV-2 E3区的穿梭质粒pVAX△E3中,构建出pVAX △E3VP2。用Sal I Nru I双酶切pVAX△E3VP2,回收含有目的基因表达盒部分,将其定向克隆入含有CAV-2 全基因组的骨架质粒pPoly2-CAV-2中,构建了重组质粒pCAV-2-FPV-VP2。Cla I Asc I酶切pCAV-2-FPV- VP2释放出重组基因组,以此转染MDCK细胞,获得了重组病毒CAV-2-VP2。该重组病毒能使MDCK细胞产生 腺病毒样细胞病变。Western blot检测证实,该重组病毒能表达具有免疫学活性的VP2蛋白。该重组病毒可以有 效地诱导免疫猫产生抗FPV和CAV-2抗体。本实验表明该重组病毒有可能成为一个FPV的疫苗株。     

Low temperature compartment formation in feline immunodeficiency virus-infected and uninfected feline kidney cells

Summary This study was to determine if feline immunodeficiency virus (FIV)-infected and uninfected Crandall feline kidney (CRFK) cells exhibited a low temperature (16°C) block in membrane trafficking between transitional endoplasmic reticulum and Golgi apparatus represented by intermediate compartment formation. Cells were cultured at different temperatures and membrane changes involving the Golgi apparatus and Golgi apparatus-associated membrane structures were monitored by electron microscopy and quantitated. With 30 min of incubation, membranes of the Golgi apparatus stack increased in amount at temperatures of 16°C and below compared to temperatures above 18°C. The increase was greatest along the major polarity axis as evidenced by an increased stack height. Neither the number of cisternae per stack nor the average stack diameter (width) was affected by temperature. The response was maximal between 15 and 30 min of low temperature treatment of the cells. Results with cells infected and uninfected with feline immunodeficiency virus were similar. The increase in stack height was due primarily to an increase of membranes at the cis face (cis Golgi apparatus network). At 18°C, membranes of the trans Golgi apparatus network accumulated suggesting that import from the cis Golgi network could proceed at this temperature, whereas exit from the trans Golgi network was still at least partially blocked. Also increased at 16°C and below were numbers of transition vesicles in the space between the Golgi apparatus and the transitional endoplasmic reticulum associated with the cis Golgi apparatus face. The results suggested interruption of the orderly flux of membranes into the Golgi apparatus at 16°C and below. Moreover, the block appeared to be reversible. Upon transfer from 16°C to 37°C, there was a time-dependent decrease in the accumulations of cis compartment membrane accompanied by a corresponding equivalent increase in the membranes of the trans Golgi apparatus compartment.     

Long-term melatonin treatment prolongs interestrus, but does not delay puberty, in domestic cats

The objective was to assess the efficacy and safety of long-term administration of melatonin (either as an implant or given orally) on interestrus intervals in domestic cats. Additionally, the effect of melatonin implants on puberty postponement was studied. For these purposes, two randomized controlled trials were conducted. In the first, 68 interestrus intervals (in 28 postpubertal queens) were studied, and in the second, 32 prepubertal female cats were used. During anovulatory interestrus intervals (27 ovulatory interestrus intervals were excluded), postpubertal cats were assigned to the following three treatments: melatonin implant 18 mg/cat SC (n = 17; MEI); melatonin tablets, 4 mg/cat/d orally until the onset of estrus (n = 12; MEO); or control (n = 12; CTL). Prepubertal females were randomly assigned to the following three treatments: melatonin 18 mg/cat sc implants at either 1.9 ± 0.3 kg (MEI-A; n = 12) or 1.5 ± 0.3 kg (MEI-B; n = 10) body weight; or control (CTL; n = 10). Interestrus intervals in postpubertal MEI, MEO, and CTL groups were 63.8 ± 5.4, 63.0 ± 5.3 and 19.2 ± 1.4 d (P < 0.05), respectively. In these groups, intervals between onset of treatment and the first estrus cycle were 51.0 ± 4.7, 50.0 ± 6.1, and 12.6 ± 1.1 d (P < 0.05). In the second experiment, neither age (MEI-A: 232.4 ± 10.5, MEI-B: 208.6 ± 13.0 and CTL: 192.4 ± 20.1 d; P > 0.1) nor body weight (P > 0.1) at puberty differed among groups. None of the cats in either study had clinically apparent side effects. We concluded that long-term melatonin treatment of domestic cats slightly prolonged interestrus intervals, but did not postpone puberty.     

Quasispecies composition and phylogenetic analysis of feline coronaviruses (FCoVs) in naturally infected cats

Quasispecies composition and tissue distribution of feline coronaviruses (FCoVs) were studied in naturally infected cats. The genomic complexity of FCoVs was investigated using single-strand conformational polymorphism (SSCP) analysis of N and ORF7b amplicons, and the evolutionary process was investigated by sequence-based phylogenetic analysis. SSCP analysis showed high heterogeneity of the FCoV genome which was correlated with the seriousness of the clinical form. The two genomic regions analysed showed different levels of variation; the N region demonstrated significant heterogeneity as compared to ORF7b. Phylogenetic analysis of the nucleotide sequences showed the clear separation of sequences analysed on the basis of virulence and geographical origin. A maximum likelihood analysis of N and ORF7b data sets showed a situation of strong heterogeneity for the N region.     

Cryopreservation of epididymal cat spermatozoa: effects of in vitro antioxidative enzymes supplementation and lipid peroxidation induction

Reactive oxygen species and lipid peroxidation reaction, causes of sperm damage, can be diminished by action of antioxidative enzymes. This study aimed to investigate effects of (1) the antioxidative enzymes; catalase, glutathione peroxidase and superoxide dismutase, on epipididymal cat sperm quality and (2) the lipid peroxidation reaction induced by a transition metal (ferrous ion (II); Fe²⁺) on sperm quality during the cryopreservation process. Epididymal spermatozoa harvested from 39 male cats were pooled and divided into 13 aliquots (n = 13). Each aliquot was resuspended with either a Tris egg yolk extender I (control; EE-I), or the Tris egg yolk extender I supplemented with 200 U/mL catalase (EE-CAT), or 10 U/mL glutathione peroxidase (EE-GPx), or 600 U/mL superoxide dismutase (EE-SOD), and then cryopreserved. After thawing, each sperm sample was subdivided into two groups; with and without lipid peroxidation induction (EE-I plus Fe²⁺, EE-CAT plus Fe²⁺, EE-GPx plus Fe²⁺ and EE-SOD plus Fe²⁺). Subjective sperm motility, membrane, and acrosome integrity were evaluated at the time of collection, after cooling, and at 0, 2, 4, and 6 h after thawing. Motility patterns assessed by computer-assisted sperm analysis (CASA), mitochondrial activity, and DNA integrity were evaluated during post-thaw incubation, whereas percentage of lipid peroxidation was detected at 0 and 6 h after thawing. The results demonstrate that catalase supplementation reduced linear motility and subjective motility immediately and 2 h after thawing (P < 0.05). Catalase supplementation, however, improved DNA integrity at 4 h (P < 0.05). Supplementation with glutathione peroxidase, compared to the control group, had a statistically significant positive effect on subjective motility at 0 and 6 h, linear motility at 6 h, mitochondrial activity at 6 h, membrane integrity at 2 and 6 h, and DNA integrity at 4 h after thawing. Although superoxide dismutase had a positive effect on sperm membrane integrity at 2 h after thawing (P < 0.05), it significantly reduced membrane integrity after cooling, linear motility at thawing, and acrosome integrity at 2 h after thawing. None of the three selected antioxidative enzymes significantly influenced acrosome integrity and none reduced the level of lipid peroxidation. Furthermore, induction of the lipid peroxidation reaction by Fe²⁺ negatively affected most of the sperm quality parameters, i.e., motility and DNA integrity, during post-thaw sperm incubation (P < 0.05). After thawing, there were, however, no significant differences between the control plus Fe²⁺ and the antioxidative enzymes supplementation plus Fe²⁺ groups. We can conclude that (1) glutathione peroxidase exhibits positive effects on post-thaw epididymal cat spermatozoa; but (2) none among the selected antioxidative enzymes could improve all sperm quality parameters; and (3) the lipid peroxidation reaction may be one cause of post-thaw epididymal sperm damage in cats, but the concentrations of antioxidative enzymes used in this study could not protect cat spermatozoa from lipid peroxidation induction.     

Effect of GnRH analogs in postnatal domestic cats

The aim of this study was to reproductively assess the clinical and hormonal effects of a GnRH agonist (AG) and an antagonist (AN) administered during the postnatal period in domestic cats. Forty-eight male and female postnatal kittens were randomly assigned to deslorelin acetate 1.6 mg subcutaneous (AG; n = 16), acyline 33 μg/100 g subcutaneous weekly for 3 months (AN; n = 16), or control (CO; n = 16) which remained untreated. The cats were followed up (behavioral observation, physical examination, fecal sexual steroid determinations, mating test, and pregnancy diagnosis) up to puberty. Puberty was delayed (weeks) in the AG animals (62.9 ± 3.5; P < 0.01) but not in the AN (15.5 ± 1.7; P > 0.05) when they were compared with CO kittens (13.4 ± 0.4). Fifteen (15/16) of the AN and CO animals, and only 11 of 16 cats of the AG group were fertile (P > 0.1). No differences were found in body weight (P > 0.1) and measurements (P > 0.1), libido (P > 0.1) and in the appearance of side effects (P > 0.1; except a pyometra in an AG female) among groups. In both AG- and AN-treated males (testosterone; P < 0.01) and females (estradiol-17β; P < 0.01) fecal hormone concentrations were lower than in CO group during the first five postnatal weeks but not later. It is concluded that the neonatal administration of these AG and AN decreased fecal sexual steroids during the first postnatal weeks causing, the agonists but not the antagonist, a significant, reversible delay in puberty appearance.     

Single residue mutation in integrase catalytic core domain affects feline foamy viral DNA integration

DD(35)E motif in catalytic core domain (CCD) of integrase (IN) is extremely involved in retroviral integration step. Here, nine single residue mutants of feline foamy virus (FFV) IN were generated to study their effects on IN activities and on viral replication. As expected, mutations in the highly conserved D107, D164, and E200 residues abolished all IN catalytic activities (3′-end processing, strand transfer, and disintegration) as well as viral infectivity by blocking viral DNA integration into cellular DNA. However, Q165, Y191, and S195 mutants, which are located closely to DDE motif were observed to have diverse levels of enzymatic activities, compared to those of the wild type IN. Their mutant viruses produced by one-cycle transfection showed different infectivity on their natural host cells. Therefore, it is likely that effects of single residue mutation at DDE motif is critical on viral replication depending on the position of the residues.     

虎源猫泛白细胞减少症病毒的分离鉴定

利用细胞培养方法从中国某虎园送检的腹泻虎肠内容物中分离出1 株细小病毒(TPV/ HT - 69),经系统的形态学、理化学、血清学试验、人工感染试验、PCR 扩增和VP2 基因序列分析,符合猫泛白细胞减少症病毒特征,证明该毒株为猫泛白细胞减少症病毒强毒。     

Exchanges of short polymorphic DNA segments predating speciation in feline major histocompatibility complex class I genes

Sequence comparisons of 14 distinct MHC class I cDNA clones isolated from species representing the three major taxonomic lineages of Felidae (domestic cat lineage, ocelot lineage, and pantherine lineage) revealed that feline MHC class I alleles have highly mosaic structures with short polymorphic sequence motifs that are rearranged between alleles of individual MHC loci, between MHC class I genes within cat species, and between homologous MHC loci in different species. The pattern of sequence variation in felids supports the role of the following factors in production and maintenance of MHC variation: (1) gradual spontaneous mutation; (2) selective pressure to conserve certain residues but also to vary in hypervariable regions, notably residues that functionally participate in antigen recognition and presentation; and (3) recombination-mediated gene exchange between alleles and between related genes. The overall amount of genetic variation observed among MHC class I genes in the Felidae family is no greater than the amount of variation within any outbred cat species (i.e., domestic cat, ocelot). The occurrence of equivalent levels of polymorphism plus the simultaneous persistence of the same sequence motifs in divergent feline species suggest that most MHC class I nucleotide site polymorphism predated species divergences. Ancient polymorphisms have been transmitted through the speciation events and modern feline MHC class I alleles were derived by recombinational exchange of polymorphic sequence motifs. Moreover, some of these sequence motifs were found in other mammalian MHC class I genes, such as classical human HLA-B5, nonclassical human HLA-E class I genes, and bovine class I genes. These results raise the prospect of an ancient origin for some motifs, although the possibility of convergence in parallel mammalian radiations cannot be excluded. Correspondence to: N. Yuhki     

Fecal estradiol-17β and testosterone in prepubertal domestic cats

The aim of this article was to describe the time course of prepubertal sexual steroids in domestic cats. Fourteen newborn kittens were followed up until puberty (physical, behavioral, and hormonal changes). Fecal testosterone [T; males] and E estradiol 17-β [E2; females] concentrations were analyzed by repeated measures ANOVA and two consecutive time windows (TWs) were used to compare changes in both male (postnatal weeks 1–4 vs. 5–14) and females (postnatal weeks 1–5 vs. 6–13). Puberty was achieved 14.3 ± 0.3 and 13.3 ± 0.4 weeks after birth in male and female cats, respectively. In both genders, during TW-1 fecal steroids concentrations were similar (males) or even higher (females) to that previously described for mature cats. Fecal T (P < 0.01) and E2 (P < 0.01) varied throughout the weeks. Differences were found when hormonal concentrations of TW-1 were compared with those of TW-2 both for male (61.4 ± 7.9 vs. 16.9 ± 2.2 ng/g; P < 0.01) and female (78.2 ± 12.5 vs. 11.2 ± 4.0 ng/g; P < 0.01) cats. It is concluded that in domestic cats there is a sexual steroid surge during the first 4 and 5 postnatal weeks in male and female animals, respectively.     

The effect of relaxin supplementation of in vitro maturation medium on the development of cat oocytes obtained from ovaries stored at 4 °C

Relaxin is a member of the insulin-like family of hormones that promotes growth in a number of reproductive tissues, including the granulosa and theca cells. Cat oocytes collected from cold-stored ovaries remain capable of maturing in vitro, but the developmental ability of the oocytes decreases after 24 h of cold storage. To improve the developmental ability of cat oocytes from cold-stored ovaries, we investigated the effect of relaxin supplementation of maturation medium on their meiotic ability and subsequent development. Cat oocytes were collected from ovaries stored at 4 °C for one day and cultured in maturation medium supplemented with different concentrations (0, 10, 20, and 40 ng/ml) of relaxin for 24 h. They were then fertilized in vitro for 12 h with frozen-thawed spermatozoa. After in vitro fertilization, the zygotes were cultured in synthetic oviduct fluid medium for 8 days. There were no significant differences in the maturation rates and glutathione contents of oocytes among the groups, irrespective of relaxin supplementation. The rate of blastocyst formation from oocytes matured with 10 ng/ml relaxin (16.0%) was higher (p < 0.05) than that from oocytes matured without relaxin (5.9%). Our findings indicate that supplementation of 10 ng/ml relaxin into maturation medium may improve blastocyst formation of cat oocytes after in vitro fertilization.     

The GnRH antagonist acyline prevented ovulation, but did not affect ovarian follicular development or gestational corpora lutea in the domestic cat

Two experiments were conducted to investigate the effects of the GnRH antagonist acyline (330 μg/kg, given sc) on ovarian follicular development and ovulation, as well as on pregnancy maintenance in domestic cats. In the first experiment, seven queens in proestrus (total of 24 proestrus periods), were randomly assigned to treatment with either acyline (ACY; n = 17) or a placebo (PLC; n = 7). All queens were mated with a fertile tomcat. In the ACY and PLC groups, cessation of estrus occurred (mean ± SEM) 7.0 ± 1.3 and 7.0 ± 1.7 d after treatment (P > 0.1), ovulation occurred in 2 of 17 and all seven estrus periods (P < 0.05), and pregnancy rates were 1 of 16 and 7 of 7 (P < 0.05), respectively. In the ACY and PLC groups, intervals from treatment to the onset of the ensuing proestrus were 18.4 ± 1.7 and 120 ± 17.2 d. In the second experiment, 14 pregnant queens were randomly allocated, according to their mating date, to treatment with acyline in early pregnancy (from 20 to 25 d, n = 3), mid pregnancy (from 26 to 45 d; n = 4), late pregnancy (> 45 d; n = 3), or injection of a placebo in early (n = 1), mid (n = 2), or late pregnancy (n = 1). Ultrasonographic assessments of the uterus were done every second day for 2 wk post treatment, and serum progesterone (P₄) concentrations were determined before treatment, and at 7 and 14 d after treatment. No pregnancies were prematurely terminated and post-treatment P₄ concentrations did not differ among treatment groups (P > 0.1). In conclusion, in the domestic cat, GnRH withdrawal by acyline prevented ovulation when given in early follicular phase (proestrus), but did not significantly affect luteal function during pregnancy.