Molecular characterization of Hepatozoon spp. infection in endangered Indian wild felids and canids

Hepatozoon species are parasites that infect a wide variety of domestic and wild animals. The objective of this study was to perform the molecular detection and characterization of Hepatozoon spp. in Asiatic lion, Indian tiger, Indian leopard, Indian wild dog, Indian domestic dog and cat based on partial 18S rRNA gene sequences from Hepatozoon spp. in the naturally infected animals. Hepatozoon spp. could be detected in blood samples of 5 out of 9 Asiatic lions, 2 out of 5 Indian tigers, 2 out of 4 Indian leopards and 2 out of 2 Indian wild dogs and, 2 out of 4 domestic cats and 2 out of 3 domestic dog samples by PCR. Sequencing of PCR amplicon and BLAST analysis of partial 18S rRNA gene sequences indicated that the Hepatozoon spp. in Asiatic lion, Bengal tiger, Indian leopard and domestic cat was Hepatozoon felis (98-99% similarity) and in the Indian wild and domestic dog the phylogenetic neighbour was Hepatozoon canis (97-100% similarity). Presence of H. felis and H. canis in both domestic and wild animals suggested that they are not host specific and the same parasite causes infection in domestic and wild felids and canids in India and from different parts of the world. To our knowledge, this is the first report on detection and molecular characterization of H. felis infection in Asiatic lions, Indian tigers, Indian leopards and H. canis in Indian wild dog. Hepatozoon spp. may be a potential pathogen and an opportunistic parasite in immuno-compromised animals and could thus represent a threat to endangered Indian wild felids and canids.     

Degenerative spinal disease in large felids.

Degenerative spinal disorders, including intervertebral disc disease and spondylosis, seldom occur in domestic cats. In contrast, a retrospective study of 13 lions (Panthera leo), 16 tigers (Panthera tigris), 4 leopards (Panthera pardis), 1 snow leopard (Panthera uncia), and 3 jaguars (Panthera onca) from the Knoxville Zoo that died or were euthanatized from 1976 to 1996 indicated that degenerative spinal disease is an important problem in large nondomestic felids. The medical record, radiographic data, and the necropsy report of each animal were examined for evidence of intervertebral disc disease or spondylosis. Eight (three lions, four tigers, and one leopard) animals were diagnosed with degenerative spinal disease. Clinical signs included progressively decreased activity, moderate to severe rear limb muscle atrophy, chronic intermittent rear limb paresis, and ataxia. The age at onset of clinical signs was 10-19 yr (median = 18 yr). Radiographic evaluation of the spinal column was useful in assessing the severity of spinal lesions, and results were correlated with necropsy findings. Lesions were frequently multifocal, included intervertebral disc mineralization or herniation with collapsed intervertebral disc spaces, and were most common in the lumbar area but also involved cervical and thoracic vertebrae. Marked spondylosis was present in the cats with intervertebral disc disease, presumably subsequent to vertebral instability. Six of the animals' spinal cords were examined histologically, and five had acute or chronic damage to the spinal cord secondary to disc protrusion. Spinal disease should be suspected in geriatric large felids with decreased appetite or activity. Radiographic evaluation of the spinal column is the most useful method to assess the type and severity of spinal lesions.     

Antibody response to vaccines for rhinotracheitis, caliciviral disease, panleukopenia, feline leukemia, and rabies in tigers (Panthera tigris) and lions (Panthera leo)

This article presents the results of a study of captive tigers (Panthera tigris) and lions (Panthera leo) vaccinated with a recombinant vaccine against feline leukemia virus; an inactivated adjuvanted vaccine against rabies virus; and a multivalent modified live vaccine against feline herpesvirus, calicivirus, and panleukopenia virus. The aim of the study was to assess the immune response and safety of the vaccines and to compare the effects of the administration of single (1 ml) and double (2 ml) doses. The animals were separated into two groups and received either single or double doses of vaccines, followed by blood collection for serologic response for 400 days. No serious adverse event was observed, with the exception of abortion in one lioness, potentially caused by the incorrect use of the feline panleukopenia virus modified live vaccine. There was no significant difference between single and double doses for all vaccines. The recombinant vaccine against feline leukemia virus did not induce any serologic response. The vaccines against rabies and feline herpesvirus induced a significant immune response in the tigers and lions. The vaccine against calicivirus did not induce a significant increase in antibody titers in either tigers or lions. The vaccine against feline panleukopenia virus induced a significant immune response in tigers but not in lions. This report demonstrates the value of antibody titer determination after vaccination of nondomestic felids.     

Prevalence of Chlamydophila felis and feline herpesvirus 1 in cats with conjunctivitis in northern Italy

The prevalence of Chlamydophila felis and feline herpesvirus 1 (FHV-1) infection in cats with conjunctivitis in northern Italy was investigated by conventional polymerase chain reaction (PCR) testing. In cats with conjunctivitis, C felis and FHV-1 were detected in 14 of 70 (20%) and in 23 of 70 (33%) animals, respectively. None of the 35 control cats were positive for C felis, whereas 7 (20%) of these cats were positive for FHV-1. Mixed infections were present in 5 of 70 cats (7%). Cats positive for C felis were significantly younger than control animals (P = .02), whereas no significant age differences were observed between FHV-1-positive cats and control cats (P = .41) or between FHV-1-positive animals and C felis-positive animals (P = .16). Cats sampled during acute-phase conjunctivitis were also investigated for the presence of C felis by conjunctival scrapings. In this acute phase, substantial agreement was found when comparing the results of the 2 methods (K = .80). The association between PCR results and conjunctivitis was evaluated for the 2 pathogens. The presence of C felis was significantly associated with conjunctivitis (P = .004), whereas the detection of FHV-1 did not significantly correlate with the clinical sign (P = .25), suggesting that, by itself. PCR is not suitable for the diagnosis of FHV-1-related conjunctivitis.     

Neutralizing antibodies against feline parvoviruses in nondomestic felids inoculated with commercial inactivated polyvalent vaccines

The virus neutralization (VN) antibody titers of serum samples from 18 individuals representing 8 carnivore species vaccinated with commercial polyvalent vaccines optimized for domestic cats containing inactivated feline panleukopenia virus (FPLV) were evaluated against canine parvovirus type 2 (CPV2). In addition, the titers among 5 individuals from 4 carnivore were evaluated against antigenic variants of feline parvoviruses; FPLV, CPV2, CPV2a, CPV2b, CPV2c, mink enteritis virus type 1 (MEV1) and MEV2. The polyvalent vaccines induced cross-reactive VN titers against antigenic variants of feline parvoviruses in nondomestic felids. However, we observed very low cross-reactive VN antibody in lions and Siberian tigers, therefore we should pay attention to CPV infections in these animals even if they were vaccinated with inactivated FPLV vaccines.     

MENISCAL OSSICLES IN LARGE NON-DOMESTIC CATS

Radiographs of the stifles of 6 species of 34 large, non-domestic cats were reviewed foremost for the presence of meniscal ossicles and then for the presence of the other potential four sesamoids. The animals in the review included 12 lions, 7 tigers, 7 cougars, 3 leopards, 3 bobcats, and 2 jaguars. Fluoroscopy, arthrography, computed tomography, necropsy, and histology were also used to evaluate the stifles of one tiger after euthanasia. Ossicles were found in the region of the cranial horn of the medial meniscus in most of the lions, tigers, leopards, and jaguars. These ossicles were found in half of the cougars but in none of the bobcats. Among the large, non-domestic cats, meniscal ossicles had been reported previously only in Bengal tigers. The lions, tigers, and leopards having meniscal ossicles appeared to have a lateral but often not a medial fabella of the gastrocnemius muscle, an observation previously unreported. Popliteal sesamoids and patellas were present in all the skeletally mature cats.     

Occurrence of Babesia felis and Babesia leo in various wild felid species and domestic cats in Southern Africa, based on reverse line blot analysis

Reverse line blot (RLB) is a hybridization assay that can be used to detect various blood parasites and differentiate between them. Results, using the RLB, showed that Babesia felis and Babesia leo occurred as single or mixed infections in various felid species, but most frequently in domestic cats and lions, respectively. Prevalence of infection in free-ranging cheetahs in Namibia was low (7, 5%), whereas 50% of free-ranging lions in South Africa and Swaziland were infected. A large number (52, 9%) of samples tested positive only for Babesia, neither B. felis nor B. leo. This could be an indication of at least one further, as yet undescribed, Babesia species in felids.     

Cats surviving natural infection with Cytauxzoon felis: 18 cases (1997-1998)

Eighteen cats surviving natural infection with Cytauxzoon felis were identified. All cats came from a limited geographic area in northwestern Arkansas and northeastern Oklahoma. Clinical signs in most cats were similar to those described for cytauxzoonosis; however, 4 cats were asymptomatic. All cases were initially diagnosed by microscopic identification of signet ring-shaped piroplasms in erythrocytes of peripheral blood smears. Four of 4 cats tested had detectable serum antibodies to C felis. Four different cats were positive by polymerase chain reaction (PCR). Partial sequencing of the PCR product from 1 cat revealed >99% homology with the reported sequence of C felis. Repeated examination of blood smears from 12 cats revealed that the erythroparasitemia was generally persistent for the duration of follow-up (3-154 days). Survival did not seem dependent on treatment, as only 1 cat was treated with a drug with potential antiprotozoal activity (imidocarb dipropionate), and 4 cats received no treatment. The findings of this study may indicate the existence of a less virulent strain of C felis.     

Cytauxzoon felis infections are present in bobcats (Lynx rufus) in a region where cytauxzoonosis is not recognized in domestic cats

This study was performed to determine the prevalence of Cytauxzoon felis (C. felis) infections in bobcats (Lynx rufus) from a region where C. felis is recognized in domestic cats, North Carolina (NC), and a region where C. felis is not recognized in domestic cats, Pennsylvania (PA). Samples from NC (n=32) were obtained post-mortem via cardiac puncture from legally trapped bobcats. Samples from PA (n=70) were collected post-mortem onto Nobuto blood collecting strips by the PA Game Commission. Each sample was tested using a C. felis specific PCR assay as well as a PCR assay targeting host DNA to rule out the presence of PCR inhibitors. Three samples were excluded due to the presence of PCR inhibitors. Thirty-three percent (10/30) of the samples from NC and 7% (5/69) of the samples from PA tested positive for the presence of C. felis. The proportion of C. felis positive bobcats from NC was significantly different than that from PA (P<0.005). Despite the lower prevalence of C. felis infections in bobcats from PA this finding is unique and indicates the potential for C. felis infections in domestic cats in the northeastern USA if the appropriate tick vectors are present. Veterinary practitioners in PA should be on alert for cytauxzoonosis in domestic cats. Further studies about the epidemiology and transmission of C. felis infections among both domestic cats and bobcats are needed.     

Use of a polymerase chain reaction assay to detect and differentiate two strains of Haemobartonella felis in naturally infected cats

OBJECTIVE: To develop a polymerase chain reaction (PCR) assay that detects and differentiates the Ohio strain of Haemobartonella felis (H. felis-OH) and the California strain of H. felis (H. felis-CA) and to apply the assay to blood samples from cats with and without suspected haemobartonellosis (suspect and control cats, respectively). SAMPLE POPULATION: 220 blood samples were examined; 82 were from suspect cats, and 138 were from control cats. PROCEDURE: A PCR assay was designed to detect and differentiate H. felis-OH and H. felis-CA. RESULTS: On the basis of PCR assay results, the overall prevalence of H. felis infection was 19.5% (43/220). Suspect cats (28.0%; 23/82) were significantly more likely than control cats (14.5%; 20/138) to be H. felis infected. Significantly greater numbers of suspect cats were H. felis-OH infected (12.2%, 9/82) or H. felis-OH and H. felis-CA infected (4.9%, 4/82) than control cats (0% [0/138] and 0.7% [1/138], respectively). Significantly more anemic cats were H. felis-OH infected (14.3%; 4/28) or H. felis-OH and H. felis-CA infected (7.1%; 2/28) than nonanemic cats (2.3% [3/128] and 0.8% [1/128], respectively). The PCR assay was more accurate than cytologic examination for detection of H. felis. CONCLUSIONS AND CLINICAL RELEVANCE: Haemobartonella felis infections are more common in cats than previously recognized. Haemobartonella felis-OH is apparently more pathogenic than H. felis-CA. The PCR assay is more accurate than cytologic examination for detection of H. felis infection and is an effective clinical tool for the detection and differentiation of both H. felis strains known to infect cats.     

Detection of persistent Cytauxzoon felis infection by polymerase chain reaction in three asymptomatic domestic cats

Repeated polymerase chain reaction (PCR) testing of 3 asymptomatic domestic cats were positive for Cytauxzoon felis DNA, suggesting persistent infection. Two cats initially presented with clinical signs consistent with acute cytauxzoonosis and, in both cases, signs of illness resolved after treatment. Parasitemia was detected in peripheral blood smears from these cats upon presentation with illness and, at subsequent follow-up appointments, in the absence of clinical illness. Polymerase chain reaction analysis was positive for C. felis from blood sampled at each time point. A third cat, a housemate of a cat fatally infected with C. felis, was preventatively treated for infection at the time of the housemate cat's death. This contact cat, having never shown signs of clinical illness consistent with cytauxzoonosis infection, had no detectable parasitemia but was positive for C. felis on repeated PCR testing. Detection of asymptomatically infected cats allows for the possibility of a yet unrecognized population of infected domestic cats that may have the capacity to serve as an additional reservoir host for C. felis, altering the currently accepted paradigm of C. felis transmission to domestic cats through bobcats as the reservoir host. In cases of very low parasitemia, more sensitive means of parasite detection, such as PCR testing, may be necessary to detect infected cats. Increased detection of asymptomatically infected cats will aid in understanding the epidemiology of C. felis infection and enhance the ability to prevent this highly fatal infectious disease of domestic cats.     

Molecular detection and characterization of Haemobartonella felis in domestic cats in Japan employing sequence-specific polymerase chain reaction (SS-PCR)

A novel PCR assay was developed in order to examine the prevalence of Haemobartonella felis (H. felis) in Japanese domestic cats and which was able to differentiate of the Ohio strain and the California strain of H. felis. Blood samples from a total of 21 cats suspected of having haemobartonellosis were examined employing a novel PCR assay and demonstrated positive results in 18 cats which was confirmed by cytological examination of blood smears. Four out of 18 positive cats (22%) were infected with the California strain, whilst the other 12 cats (67%) were infected with the Ohio strain and two animals (11%) were infected with both strains. As most of the cats with moderate to severe anemia were infected with the Ohio strain, it is suggested that the most prevalent strain of H. felis in Japanese domestic cats might be the Ohio strain. In the present study, it was thought that molecular detection and characterization of H. felis may provide valuable information regarding the severity and prognosis of this illness.     

Development and evaluation of a PCR assay for the detection of Cytauxzoon felis DNA in feline blood samples

Cytauxzoonosis is an emerging tick borne infectious disease of domestic cats in the United States, caused by the organism Cytauxzoon felis (C. felis). In naturally infected domestic cats the disease is almost always fatal. Currently there are no commercially available molecular or serologic tests to facilitate the antemortem diagnosis of C. felis infection. Clinical and pathological diagnosis of cytauxzoonosis is based on microscopic identification of parasites in tissues or on blood smears. We have developed and evaluated the sensitivity and specificity of a polymerase chain reaction (PCR) based assay for the diagnosis of C. felis infections in feline blood samples. The assay is sensitive enough to detect one copy of a cloned fragment of the C. felis 18S rRNA gene. This PCR assay can be used for the rapid clinical diagnosis of cytauxzoonosis and for epidemiological studies that will better define the geographic distribution of C. felis infection in cats.     

Prevalence of Rickettsia felis DNA in the blood of cats and their fleas in the United States

Rickettsia felis is associated with fever, headache, myalgia, and macular rash in some infected humans and has been detected in the cat flea (Ctenocephalides felis) in many countries around the world. While some naturally exposed cats have been assessed for antibodies against R felis, to our knowledge, no one has reported use of polymerase chain reaction (PCR) to attempt to amplify R felis DNA from client-owned cats and the fleas collected from them. In this study, we assayed 92 pairs of cat blood and flea extracts from Alabama, Maryland and Texas, using PCR assays that amplify a region of the citrate synthase gene (gltA) and the outer membrane protein B gene (ompB). Of the 92 pairs, 62 of 92 (67.4%) flea extracts and none of the cat blood samples were positive for R felis DNA.     

Use of enrofloxacin for treatment of large-form Haemobartonella felis in experimentally infected cats

OBJECTIVE: To compare treatment with enrofloxacin and doxycycline with no treatment in cats experimentally infected with Haemobartonella felis. DESIGN: Prospective case-control study. ANIMALS: 16 cats. PROCEDURE: Cats were inoculated with large-form H. felis from a chronically infected donor. Cats were assigned to 1 of 4 treatment groups: doxycycline (5 mg/kg [2.3 mg/lb], p.o., q 12 h), low-dose enrofloxacin (5 mg/kg, p.o., q 24 h), high-dose enrofloxacin (10 mg/kg [4.5 mg/lb], p.o., q 24 h), and an untreated control group. Clinical signs, Hct, blood smears, and a polymerase chain reaction (PCR) assay were used to monitor progression of the infection. RESULTS: All cats were confirmed to be infected with H. felis via blood smear evaluations and PCR assay results. Treatment had no effect on Hct during the intratreatment period, but Hct values were significantly greater in the low-dose enrofloxacin group, compared with the control group, during the posttreatment period. During the intratreatment period, H. felis organism counts per 1,000 RBC in the doxycycline treatment and the high-dose enrofloxacin treatment groups decreased at a significantly faster rate than those in the control group. In the posttreatment period, organism counts in the doxycycline treatment group and the low- and high-dose enrofloxacin groups decreased at significantly faster rates than counts in the control group. There was no significant effect of treatment on the number of positive PCR assay results. Two cats treated with enrofloxacin and 1 cat treated with doxycycline completely cleared the H. fe is organism despite presumed immunosuppression caused by glucocorticoids. CONCLUSIONS AND CLINICAL RELEVANCE: Results support the hypothesis that enrofloxacin has anti-H. felis effects.     

Haematological reference values for adult pumas, lions, tigers, leopards, jaguars and cheetahs

Normal haematological values and fibrinogen levels were obtained from a number of healthy adult Felidae in the collection of the Zoological Society of London. The group comprised 29 pumas (Felis concolor), 32 lions (Panthera leo), 27 tigers (P tigris), 19 leopards (P pardus), 18 jaguars (P onca) and 22 cheetahs (Acinonyx jubatus). The values provided a basis for identifying abnormalities in the blood of sick individuals of these species and for undertaking interspecies comparisons.     

Inoculation of two genotypes of Hemobartonella felis (California and Ohio variants) to induce infection in cats and the response to treatment with azithromycin

OBJECTIVES: To describe clinical and laboratory findings associated with cats experimentally infected by inoculation with the 2 recognized genotypes of Hemobartonella felis (small variant, Hfsm; large variant, Hflg) and to determine the response of cats to treatment with azithromycin. ANIMALS: 18 young adult domestic shorthair cats of both sexes. PROCEDURES: Cats were inoculated with H felis and monitored weekly, using CBC counts and a polymerase chain reaction (PCR) designed to detect both genetic variants of H felis. Beginning 26 days after inoculation, 11 cats were administered azithromycin (15 mg/kg of body weight, PO, q 12 h, for 7 days). RESULTS: Inoculation resulted in coinfection with Hflg and Hfsm, and both variants were detected by PCR. Clinical abnormalities and anemia were most severe in Hflg- and dual-infected cats. Results of PCR and CBC were positive for H felis in 112/112 (100%) and 42/112 (37.5%), respectively, samples collected after inoculation. Administration of azithromycin had little effect on clinical variables, including anemia. All cats, regardless of treatment with azithromycin, had positive results for the PCR at the end of the study period. CONCLUSIONS AND CLINICAL RELEVANCE: In these cats, Hflg was more pathogenic than Hfsm, and coinfection with both variants was detected. Results of the PCR were superior to results of CBC for detecting infection with H felis. Azithromycin administered at the dose and duration reported here was not efficacious for the treatment of cats with hemobartonellosis.     

Cytologic findings, and feline herpesvirus DNA and Chlamydophila felis antigen detection rates in normal cats and cats with conjunctival and corneal lesions

Samples were collected from 36 cats with feline herpesvirus (FHV-1)-related ocular disease (conjunctivitis, epithelial or stromal keratitis, or corneal sequestration), and 17 cats without ocular changes. Corneoconjunctival swabs, scrapings and biopsies were tested in various combinations for presence of FHV-1 DNA using single round (sr) polymerase chain reaction (PCR) and nested PCR (nPCR). Additional swabs from the inferior conjunctival fornix were tested by enzyme-linked immunosorbent assay for Chlamydophila felis antigen. Cytologic evaluation was carried out on conjunctival (cats with conjunctivitis) and corneal (cats with keratitis) cytobrush preparations. FHV-1 DNA was detected by PCR in 14 (39%) cats with ocular disease and 1 (6%) of the control group. Agreement between srPCR and nPCR results was significant (P < 0.01). FHV-1 DNA was detected in 3/7 cats with conjunctivitis, 5/6 cats with epithelial keratitis, 3/11 cats with stromal keratitis, and 3/12 cats with corneal sequestration. There was a significant association (P = 0.0027) between viral presence and epithelial keratitis. However, no significant association was found between viral presence and conjunctivitis (P = 0.059), stromal keratitis (P = 0.15), or corneal sequestration (P = 0.18). With respect to FHV-1 DNA detection, intersample agreement was significant (P < 0.03). No sampling technique seemed more likely than another to harvest detectable viral DNA, except for cats with corneal sequestrum in which viral DNA was not detected using corneoconjunctival swabs. FHV-1 DNA was detected in 6/9 samples with intranuclear inclusion bodies and in 6/7 cats with eosinophils on cytologic examination. All samples tested negative for C. felis antigen.     

Prevalence of feline herpesvirus 1, Chlamydophila felis, and Mycoplasma spp DNA in conjunctival cells collected from cats with and without conjunctivitis

OBJECTIVE: To use PCR assays to determine the prevalence of feline herpesvirus 1 (FHV-1), Chlamydophila felis, and Mycoplasma spp DNA in conjunctival cells collected from cats with and without conjunctivitis; to compare results of conventional and real-time fluorogenic PCR assays for amplification of FHV-1 DNA; and to determine whether copy numbers of FHV-1 DNA are correlated with conjunctivitis. ANIMALS: 55 cats with active conjunctivitis, 39 healthy cats that never had conjunctivitis, and 32 cats with a history of conjunctivitis that had been resolved for at least 3 months. PROCEDURES: Samples were obtained by rolling cotton-tipped applicators on the ventral conjunctiva of awake cats treated topically with proparacaine. The DNA was extracted from the swab specimens and assessed in PCR assays to detect DNA of FHV-1 (fluorogenic PCR assay and conventional PCR assay), Mycoplasma spp (conventional PCR assay), and C felis (conventional PCR assay). RESULTS: Overall prevalence rates of FHV-1, C felis, and Mycoplasma spp as assessed by the conventional PCR assays were 6.7%, 3.2%, and 9.6%, respectively. Percentage concordance between conventional PCR and fluorogenic PCR assays for FHV-1 was 92.5%. There were no significant differences among the 3 groups of cats for the mean copy number of FHV-1 divided by the copy number of glyceraldehyde-3-phosphate dehydrogenase. CONCLUSIONS AND CLINICAL RELEVANCE: Mycoplasma spp were the most prevalent organism detected and was associated with conjunctivitis. This study could not confirm that there are increased copy numbers of FHV-1 DNA in cats with conjunctivitis, compared with the copy numbers for cats without conjunctivitis.     

Prevalence of Rickettsia species antibodies and Rickettsia species DNA in the blood of cats with and without fever

Rickettsia species antibodies have been detected in some cats but it is unknown whether infected cats develop clinical signs. The prevalence of Rickettsia species deoxyribonucleic acid (DNA) in blood from clinically ill cats has not been determined. The objective of this study was to determine if cats with fever (body temperature >or=102.5 degrees F [39.2 degrees C]) were more likely to have evidence of rickettsial infection than healthy, age-matched, control cats with a body temperature<102.5 degrees F. Rickettsia species polymerase chain reaction (PCR) assays were performed to detect rickettsial DNA extracted from blood (71 paired samples), indirect immunofluorescence assays (IFA) were performed to detect serum antibodies against Rickettsia felis (90 paired samples) and Rickettsia rickettsii (91 paired samples), and the results between pairs were compared. All samples were negative for Rickettsia species DNA. More cats with fever were seropositive for R felis or R rickettsii than control cats, but results were not statistically significant. Results of this pilot study failed to show an association between Rickettsia species DNA or Rickettsia species antibodies and fever.