Lynn Guptill

Experimental Infection of Young Specific Pathogen-Free Cats with Bartonella henselae

Eighteen 12-week-old specific pathogen-free cats, blood culture- and serum antibody-negative for Bartonella henselae, were randomly allocated to groups and were intravenously inoculated with 10(10) (group 1), 10(8) (group 2), or 10(6) (group 3) B. henselae or with saline (group 4) or were not inoculated (group 5). Cats were humanely killed at 2, 4, 8, 16, and 32 weeks after inoculation. All B. henselae-inoculated cats were bacteremic by 2 weeks after infection. Bacteremia persisted until 32 weeks after infection in 1 cat. Cats in groups 1 and 2 had fever (>39.7 degrees C) and partial anorexia by 2 weeks after infection that lasted 2-7 days. All infected cats had Bartonella-specific IgM and IgG serum antibodies and lymphocyte blastogenic responses. Histopathologic lesions were observed in multiple organs of infected cats through 8 weeks after infection. Cats were readily infected with B. henselae by intravenous inoculation, developed histopathologic lesions that apparently resolved, and developed B and T lymphocyte responses to infection.

Time Trends and Risk Factors for Diabetes Mellitus in Dogs: Analysis of Veterinary Medical Data Base Records (1970–1999)

The objectives of the study were to identify recent trends in the prevalence of diabetes mellitus (DM) in dogs and to identify host risk factors. Veterinary Medical Data Base (VMDB) electronic records of 6860 dogs with a diagnosis of DM (VMDB code 870178500) between 1970 and 1999 were evaluated to determine time trends. Records of 6707 dogs with DM and 6707 frequency matched dogs with any diagnosis other than DM from the same teaching hospitals in the same year, selected as controls, were evaluated for risk factor analysis. The prevalence of DM in dogs presented to veterinary teaching hospitals increased from 19 cases per 10,000 admissions per year in 1970 to 64 cases per 10,000 in 1999, while the case-fatality rate decreased from 37% to 5%. The hospital prevalence of DM was consistently greater over time in older compared with younger dogs with the highest prevalence occurring in dogs 10-15 years of age. Dogs weighing <22.7 kg had a significantly (P<0.001) greater risk of DM compared with heavier dogs. Female dogs had an increased risk of DM compared with males (P<0.001).

Time trends and risk factors for diabetes mellitus in cats presented to veterinary teaching hospitals

Veterinary Medical Data Base records of cats with diabetes mellitus (DM) from 1970 through 1999 were reviewed to identify trends in hospital prevalence of DM and potential host risk factors. Hospital prevalence increased from eight cases per 10,000 in 1970 to 124 per 10,000 in 1999 (P < 0.001). Case fatality percent at first visit decreased from 40% to 10% (P < 0.001). Hospital prevalence increased in all age groups (P < 0.002). There was no apparent seasonal pattern in hospital prevalence. Significant risk factors included male gender, increasing age for both genders (P < 0.001), increasing weight for males (P < 0.001), and mixed vs pure breed for females (P = 0.006).

Evidence of reproductive failure and lack of perinatal transmission of Bartonella henselae in experimentally, infected cats

Five female specific pathogen-free (SPF) cats inoculated intradermally with B. henselae and bacteremic for 4 weeks, and one cat inoculated with 0.9% NaCl, were bred with uninfected SPF male cats. The uninfected female became pregnant with one breeding, while three infected cats became pregnant 1-12 weeks later, after repeated breedings. Two infected females either did not become pregnant or maintain pregnancies despite repeated breedings. Infected cats produced anti-B. henselae IgM and IgG antibodies. Fetuses and kittens of infected cats were not infected and did not produce anti-B. henselae antibodies. Male cats bred with infected females did not become infected or seroconvert. Maternal anti-B. henselae IgG antibodies detected in sera of kittens 2 weeks post-partum were no longer detectable 10 weeks post-partum. These findings suggest that B. henselae causes reproductive failure in female cats, but is not transmitted transplacentally, in colostrum or milk, or venereally. Infected cats immunosuppressed with methylprednisolone acetate after their kittens were weaned had no detectable bacteria in tissues, suggesting that they were no longer infected.

Canine leptospirosis, United States, 2002-2004.

The proportion of positive Leptospira microscopic agglutination tests for 23,005 dogs significantly increased from 2002 to 2004 (p<0.002) regardless of the positive cutoff titer used and was highest (p<0.05) for serovars Autumnalis and Grippotyphosa. The strongest positive serologic correlation (r = 0.72) was between serovars Autumnalis and Pomona.

American Association of Feline Practitioners 2006 Panel report on diagnosis, treatment, and prevention of Bartonella spp. infections:

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Detection of antibodies against Leptospira serovars via microscopic agglutination tests in dogs in the United States, 2000–2007

OBJECTIVE To use results of microscopic agglutination tests (MATs) conducted at a commercial veterinary diagnostic laboratory to determine temporal and demographic distributions of positive serologic test results for leptospirosis in dogs and identify correlations among results for various Leptospira serovars. DESIGN Serosurvey. STUDY POPULATION MAT results for 33,119 canine serum samples submitted to a commercial veterinary diagnostic laboratory from 2000 through 2007. PROCEDURES Electronic records of MAT results for dogs were obtained from a veterinary diagnostic laboratory. Seropositivity for antibodies against Leptospira serovars was determined by use of a cutoff titer of >or=1:1,600 to reduce the possible impact of postvaccinal antibodies on results. Correlations between results for all possible pairs of serovars were calculated by ordinal ranking of positive (>or=1:100) antibody titer results. RESULTS 2,680 samples (8.1%; 95% confidence interval [CI], 7.8% to 8.4%) were seropositive for antibodies against Leptospira serovars. The highest percentage of positive MAT results was for the year 2007 (10.2%; 95% CI, 9.5% to 10.9%) and for the months of November and December during the study period. Antibodies were most common against serovars Autumnalis, Grippotyphosa, Pomona, and Bratislava. Seroprevalence of leptospirosis was lowest for dogs>10 years of age but was similar across other age strata. CONCLUSIONS AND CLINICAL RELEVANCE Leptospirosis can affect dogs of small and large breeds and various ages. Although an increase in proportions of positive MAT results was evident in the fall, monthly and annual variations suggested potential exposure in all months. Because of the limitations of MAT results and the limited number of serovars used in the test, bacterial culture should be used to identify infective Leptospira serovars.

Immune response of neonatal specific pathogen-free cats to experimental infection with Bartonella henselae

The purpose of this study was to determine whether neonatal cats develop and maintain a persistent bacteremia for longer than do adult cats with a normal mature immune system, and whether neonatal cats are susceptible to infection with Bartonella henselae by oral inoculation. Neonatal specific pathogen-free (SPF) cats were inoculated with B. henselae intradermally (n = 4) or orally (n = 5) or with 0.9% NaCl (n = 2). Blood was collected periodically through 16 weeks post-inoculation (PI) for serology, bacteriology and complete blood count. Cats inoculated orally or intradermally at 3-5 days of age were bacteremic through 12-16 weeks PI, similar to what is documented for adult cats inoculated intradermally or intravenously. One cat inoculated at age 2 weeks was bacteremic through 10 weeks PI; the other was not bacteremic. Intradermally inoculated neonatal cats produced serum IgG antibodies to B. henselae but orally inoculated neonatal cats did not. Infected cats with and without serum IgG antibodies to B. henselae became blood-culture negative simultaneously, suggesting that IgG is not required to clear bacteremia.

Spatial and spatio-temporal clustering of overall and serovar-specific Leptospira microscopic agglutination test (MAT) seropositivity among dogs in the United States from 2000 through 2007.

Leptospirosis is a re-emerging disease of dogs in the United States (U.S.). This paper reports the findings of a retrospective study conducted to determine if seroreactivity to Leptospira microscopic agglutination test (MAT) among dogs in the U.S. clustered in space and time. The study utilized canine sera submitted to a commercial laboratory for leptospiral MAT from January 2000 through December 2007. There were 31,869 serum samples submitted by veterinarians from 3156 zip code locations across the U.S. Results of MAT were considered positive at titers of > or = 1:1600. Spatial and spatial-temporal scan statistics were used to identify statistically significant clusters of seroreactivity to Leptospira (overall and individual serovars) using recorded test request dates and locations of the centroid of the zip code reported for each serum sample. There were 2469 positive MAT results with a titer > or = 1:1600 to at least one of seven Leptospira serovars. Two relevant spatial clusters of 26.3 and 246.5 km radius were identified (P=0.001). The primary cluster was located in the northeastern part of Illinois including Chicago and surrounding areas (232 [14.4%] of 1612 MAT positive; RR=1.95). The secondary cluster covered the central part of Texas (292 [12.62%] of 2314 MAT positive; RR=1.71). Eight space-time clusters of overall MAT positivity were identified (29-335 km radius; P=0.001-0.048 and RR=3.98-24.69) that covered different geographic locations for different time points. Spatial and space-time clusters for individual serovars were also identified for six serovars: eight each of Grippotyphosa and Pomona, seven of Bratislava, five of Autumnalis, and three each of Icterohaemorrhagiae and Canicola. In conclusion Leptospira seropositivity in dogs tended to have distinctive clusters in space and space-time. Most of the space-time clusters of overall Leptospira MAT seropositivity were associated with cluster events for individual serovars. Further investigation is warranted to explain individual serovar clusters detected in this study, as a complex interaction of incidental host, environment and reservoir host may be responsible for the occurrence of these serovar clusters.

Antibacterial Activity of Novel Cationic Peptides against Clinical Isolates of Multi- Drug Resistant Staphylococcus pseudintermedius from Infected Dogs

Staphylococcus pseudintermedius is a major cause of skin and soft tissue infections in companion animals and has zoonotic potential. Additionally, methicillin-resistant S. pseudintermedius (MRSP) has emerged with resistance to virtually all classes of antimicrobials. Thus, novel treatment options with new modes of action are required. Here, we investigated the antimicrobial activity of six synthetic short peptides against clinical isolates of methicillin-susceptible and MRSP isolated from infected dogs. All six peptides demonstrated potent anti-staphylococcal activity regardless of existing resistance phenotype. The most effective peptides were RRIKA (with modified C terminus to increase amphipathicity and hydrophobicity) and WR-12 (α-helical peptide consisting exclusively of arginine and tryptophan) with minimum inhibitory concentration50 (MIC50) of 1 µM and MIC90 of 2 µM. RR (short anti-inflammatory peptide) and IK8 “D isoform” demonstrated good antimicrobial activity with MIC50 of 4 µM and MIC90 of 8 µM. Penetratin and (KFF)3K (two cell penetrating peptides) were the least effective with MIC50 of 8 µM and MIC90 of 16 µM. Killing kinetics revealed a major advantage of peptides over conventional antibiotics, demonstrating potent bactericidal activity within minutes. Studies with propidium iodide and transmission electron microscopy revealed that peptides damaged the bacterial membrane leading to leakage of cytoplasmic contents and consequently, cell death. A potent synergistic increase in the antibacterial effect of the cell penetrating peptide (KFF)3K was noticed when combined with other peptides and with antibiotics. In addition, all peptides displayed synergistic interactions when combined together. Furthermore, peptides demonstrated good therapeutic indices with minimal toxicity toward mammalian cells. Resistance to peptides did not evolve after 10 passages of S. pseudintermedius at sub-inhibitory concentration. However, the MICs of amikacin and ciprofloxacin increased 32 and 8 fold, respectively; under similar conditions. Taken together, these results support designing of peptide-based therapeutics for combating MRSP infections, particularly for topical application.