Enrichment-toy trauma in a New Zealand White rabbit.

An injury was caused by an enrichment toy (a whiffle ball, which is a perforated hollow ball made of hard plastic) that led to its removal from the rabbit enrichment program. Manipulata and food treats form the basis of the Yale rabbit enrichment program. All singly housed rabbits are given toys such as balls, chains, wood blocks, PVC tubing, Nylabones, and corrugated plastic tunnels. Before they are used, all potential enrichment devices are reviewed for safety and potential veterinary problems. The whiffle ball had been considered safe because it was made of hard non-toxic plastic, had no sharp edges, was too large to be swallowed or inhaled, and was judged too sturdy to be broken by the rabbits. However, the ball became lodged in the incisors of an adult female New Zealand White rabbit, preventing her from eating or drinking for 12 h and causing marked trauma to her gums. Removal of the ball necessitated anesthetizing the rabbit and using bone cutters to cut away the ball. Ideally, environmental enrichment should increase species-specific normal behavior and minimize stereotypies and self- and conspecific-directed abusive behavior. This case illustrates that safety assessments for an enrichment device must include both the inherent properties of the device and the risks if the toy is misused or damaged. Considerations for safety assessment are discussed.

Ancylostoma caninum anticoagulant peptide blocks metastasis in vivo and inhibits factor Xa binding to melanoma cells in vitro.

We evaluated the in vivo anti-metastatic activity of recombinant Ancylostoma caninum Anticoagulant Peptide (rAcAP), a potent (Ki = 265 pM) and specific active site inhibitor of human coagulation factor Xa originally isolated from bloodfeeding hookworms. Subcutaneous injection of SCID mice with rAcAP (0.01-0.2 mg/mouse) prior to tail vein injection of LOX human melanoma cells resulted in a dose dependent reduction in pulmonary metastases. In order to elucidate potential mechanisms of rAcAP's anti-metastatic activity, experiments were carried out to identify specific interactions between factor Xa and LOX. Binding of biotinylated factor Xa to LOX monolayers was both specific and saturable (Kd = 15 nM). Competition experiments using antibodies to previously identified factor Xa binding proteins, including factor V/Va, effector cell protease receptor-1, and tissue factor pathway inhibitor failed to implicate any of these molecules as significant binding sites for Factor Xa. Functional prothrombinase activity was also supported by LOX, with a half maximal rate of thrombin generation detected at a factor Xa concentration of 2.4 nM. Additional competition experiments using an excess of either rAcAP or active site blocked factor Xa (EGR-Xa) revealed that most of the total factor Xa binding to LOX is mediated via interaction with the enzyme's active site, predicting that the vast majority of cell-associated factor Xa does not participate directly in thrombin generation. In addition to establishing two distinct mechanisms of factor Xa binding to melanoma, these data raise the possibility that rAcAP's antimetastatic effect in vivo might involve novel non-coagulant pathways, perhaps via inhibition of active-site mediated interactions between factor Xa and tumor cells.

Experimental Borrelia burgdorferi infection in Peromyscus leucopus.

We evaluated the susceptibility of laboratory-reared adult and infant white-footed mice (Peromyscus leucopus) to a known pathogenic isolate of Borrelia burgdorferi (N40). Two-month-old and 3-day-old Peromyscus were inoculated intradermally with 10(6) to 10(7) spirochetes. At 21 days for adults or 30 days for infants post inoculation, mice were killed, and tissues were cultured for spirochetes and examined microscopically. Based on serology and culture, adult mice became infected but did not have any gross or microscopic lesions. Mice inoculated as infants became infected, and also developed carditis and multifocal arthritis. Contact transmission between inoculated infants and their naive mothers was not observed. Age at inoculation appeared to be a critical factor in inducing Lyme borreliosis lesions in Peromyscus leucopus, as in other species.

Long-term study of cell-mediated responses to Borrelia burgdorferi in the laboratory mouse.

Borrelia burgdorferi infection of disease-susceptible (C3H) and -resistant (BALB) mice resulted in impaired proliferation to both T- and B-cell mitogens up to 30 days after inoculation. Interleukin-2 and -4 production was also impaired, paralleling the T-cell response to concanavalin A. Impaired lymphocyte proliferation could not be attributed to diminished numbers of T or B cells and was found to depend on the lymphoid organ (spleen or lymph node) examined. Prostaglandin production accounted for part of this immune dysfunction. Attempts to assess antigen-specific proliferation to B. burgdorferi were inconsistent, and delayed-type hypersensitivity responses were not detected. Adoptive transfer of T-enriched cells from chronically infected donors failed to prevent infection and disease development in recipient C3H mice. The current study emphasizes caution in the study of B. burgdorferi antigen-specific assays and argues against the role of a vigorous T-cell response in Lyme borreliosis in infected laboratory mice.

Synthetic parathyroid hormone-like protein (1-74) is anabolic for bone in vivo.

Parathyroid hormone-related protein (PTHRP) has recently been purified from human tumors associated with the syndrome of humoral hypercalcemia of malignancy. The gene encoding PTHRP has been cloned, and based on predicted amino acid sequence, polypeptides comprising the first 36 [36Tyr(1-36) PTHRP amide] and 74 [(1-74)PTHRP] amino acids have been synthesized. Human (h) PTHRP (1-36) and (1-74) are potent bone-resorbing agents, and are catabolic for bone in vivo when given continuously at high doses. Bovine parathyroid hormone (bPTH) (1-34) is also catabolic for bone at high dose levels, but when given in low doses for weeks to months, it is anabolic. Although PTHRP possess several PTH-like properties in bone, hPTHRP (1-34) is reported to be only weakly anabolic in vivo. As polypeptide length influences PTHRP action, we evaluated hPTHRP(1-74) as an anabolic agent for bone in vivo. Twenty-four 4-week-old male Sprague-Dawley rats were given daily subcutaneous injections of hPTHRP(1-74) (1 and 2 nmol/100 g body weight, bw), bPTH(1-34) (4 nmol/100 g bw) or vehicle. Rats were sacrificed on day 12, and serum calcium, phosphorus, and 1,25 dihydroxyvitamin D and femoral bone dry weight, calcium content, and hydroxyproline content were measured. Serum calcium and phosphorus were equivalent in all groups. A significant increase in dry bone weight was observed in both PTHRP-treated groups compared with controls. PTHRP also caused a significant, dose-dependent increase in bone calcium and hydroxyproline content. Results of these studies indicate that PTHRP (1-74) is anabolic for bone in vivo when administered at low-dosage levels for a prolonged period.

Lyme borreliosis in laboratory animals: effect of host species and in vitro passage of Borrelia burgdorferi.

The susceptibility of several common laboratory animal species to a known pathogenic isolate of Borrelia burgdorferi (N40) was evaluated following intraperitoneal (ip) inoculation of 10(6-8) spirochetes into 3-day-old Lewis rats, CD-1 mice, Syrian hamsters, and 3-week-old American Dutch rabbits. At 30 days, tissues were cultured for spirochetes and examined histologically. All species developed multisystemic infection as well as arthritis and carditis, but disease was most severe in rats and mice. In order to evaluate the effect of in vitro passage on the pathogenicity of B. burgdorferi, 3-day-old Lewis rats were inoculated ip with borreliae passaged in culture 2, 5, 11, 17, 21, 26, and 31 times, and evaluated at 30 days by culture, histology, and ELISA antibody titers. Based upon these parameters, B. burgdorferi (N40) lost its virulence at 17-21 passages. This study demonstrated that B. burgdorferi was infectious for infant rats, mice, hamsters, and 3-week-old rabbits, although pathogenicity was modulated by host species and the in vitro passage history of the spirochete. Of the 4 laboratory animal species evaluated in this study, rats and mice appear to have the most potential for further use as animal models of Lyme disease.

Lyme borreliosis in selected strains and ages of laboratory mice.

The susceptibility of laboratory mice to Borrelia burgdorferi was evaluated for selected genotypes and ages. C3H/He, SWR, C57BL/6, SJL, and BALB/c mice inoculated at age 3 days developed uniformly severe polyarthritis at 30 days after intraperitoneal inoculation. Mice inoculated at age 3 weeks also developed polyarthritis, but severity was influenced by genotype, with C3H/He and SWR mice the most severely affected. Susceptible strains developed higher IgG ELISA antibody titers to B. burgdorferi than did resistant mice. Adult (12 weeks) C3H/He mice were also susceptible, but arthritis was not as severe as in those inoculated at age 3 weeks. SKH (hairless) mice developed polyarthritis but not skin disease when inoculated intradermally. Carditis occurred frequently among C3H/He, BALB/c, and hairless mice and in some SWR mice but not in C57BL/6 or SJL mice. This study demonstrates that severity of Lyme borreliosis is age- and genotype-dependent and that laboratory mice are a potentially valuable model.

Experimental chronic Lyme borreliosis in Lewis rats.

The course of Lyme borreliosis in LEW/N rats inoculated intraperitoneally as infants with 10(6) Borrelia burgdorferi was followed for 360 days. Spirochetes were detected in the blood through 30 days, in the brain through 60 days, and persisted in the spleen, liver, kidneys and articular tissue through 360 days. Acute exudative arthritis, tendonitis, and bursitis were evident in multiple joints by day 30. Arthritis regressed thereafter but capsular fibrosis and lymphoplasmacytic infiltrates persisted throughout the study. Several rats developed exacerbations of acute arthritis within days 180-360, a pattern similar to that encountered in human Lyme disease. Rats had a high prevalence of nonsuppurative myocarditis and vasculitis during days 90-360. Spirochetes were visualized by microscopy in joints and other tissues during the first month of infection, but were seen only sporadically thereafter. All rats seroconverted to B. burgdorferi by day 30. IgM titers persisted and IgG titers rose progressively through day 360. Immunoblots revealed IgM reactivity to a single 41 kDa protein until 360 days, when reactivity to a 60 kDa protein emerged. IgG reactivity occurred against progressively more proteins with time, indicating continued antigenic stimulation. Chronic and recurrent arthritic lesions and myocardial involvement suggest that the rat is a reliable model for further investigation.

An animal model for Lyme arthritis.

A model of Lyme arthritis has been developed in laboratory rats. Intraperitoneal inoculation of a low-passage tick isolate of B. burgdorferi into neonatal and weanling LEW/N rats resulted in multisystemic infection and arthritis. Spirochetes were isolated from blood, liver, kidney, spleen, brain, and joints of inoculated rats. Arthritis, associated with the presence of spirochetes, developed in multiple joints by day 14 and persisted through day 90 after inoculation. Arthritic lesions resembled those found in human Lyme disease lesions. Lesions were not found in other organs, although spirochetes were present. Neonatal F344 and SD rats were also susceptible to infection and induction of arthritis. Three different isolates of B. burgdorferi were shown to be pathogenic. Pathogenicity of one isolate was retained after at least 11 in vitro passages. Formalin-killed spirochetes were not pathogenic. Other features of the Lyme disease complex have yet to be seen in the rat, but long-term studies are required to completely define the rat model. This highly reproducible model should allow in-depth studies on the pathogenetic mechanisms of this important human disease.

Experimental Corynebacterium kutscheri infection in rats: bacteriology and serology.

In a preliminary study, hydrocortisone-treated rats developed pseudotuberculosis when challenged with 6.2 X 10(5) to 3.1 X 10(7) colony forming units of Corynebacterium kutscheri by intranasal, intragastric, or subcutaneous inoculation. Oronasal exposure was selected as a likely natural route to further study inapparent infection. In Study 1, 50 rats received 1.2 X 10(5) colony forming units and various tissues were cultured at intervals to 12 weeks post-inoculation. At each interval, C. kutscheri was regularly isolated from submaxillary lymph nodes, but isolation was sporadic from other sites. In Study 2, 17 out of 21 rats given 1.2 X 10(5) colony forming units and killed weekly for 6 weeks had 2.0 X 10(2) to 1.8 X 10(5) colony forming units of C. kutscheri in oral washes, and 16 rats had 2.0 X 10(2) to 1.0 X 10(5) colony forming units in submaxillary lymph nodes, Serum antibody to C. kutscheri using both microagglutination and indirect immunofluorescence was first detected in some rats by 2 weeks, and in all rats at subsequent intervals. There was a significant (P less than 0.001) positive correlation (r = 0.93) between serum antibody titers and the duration of infection.