Ling-Ling Chueh

Genetic diversity and correlation with feline infectious peritonitis of feline coronavirus type I and II: A 5-year study in Taiwan

Abstract The outcomes of feline coronavirus (FCoV) infection vary greatly from asymptomatic or mild enteric infection to fatal feline infectious peritonitis (FIP). On the basis of in vitro neutralization tests, FCoVs can be divided into two serotypes. To explore the correlation between different types of FCoV and FIP, clinical specimens collected from 363 naturally infected cats during 2003–2007 were analyzed. Amplification of a portion of the S gene from the FCoV was performed and a total of 222 cases were differentiated. Among them, 197 (88.7%) cats were type I-positive, 13 (5.9%) were type II-positive, and 12 (5.4%) were positive for both types. Irrespective of the predominance of type I FCoV infection in Taiwan, type II FCoV demonstrated a significantly higher correlation with FIP (p <0.01). Analysis of partial S gene sequences of the local type I and II FCoVs strains revealed that type I viruses were more genetically divergent (6.2–11.7%) than type II viruses (0.6–3.2%) within the 5-year study period. The higher genetic diversity of type I FCoVs might be due to the larger infected cat population and to the long period of viral persistence in asymptomatic cats in comparison to type II viruses.

Field strain feline coronaviruses with small deletions in ORF7b associated with both enteric infection and feline infectious peritonitis

Feline coronavirus (FCoV) varies greatly from causing subclinical or mild enteric infections to fatal feline infectious peritonitis (FIP). The open reading frame (ORF) 7b of FCoV has been speculated to play a determining role in virulence as deletions were found to be associated with avirulent viruses. To further clarify the correlation between this gene and FIP, clinical samples from 20 cats that had succumbed to wet-type FIP and 20 clinically healthy FCoV-infected cats were analysed. The ORF7b from the peritoneal/pleural effusions of FIP cats and from the rectal swabs of healthy cats was amplified. Of the 40 FCoVs analysed, 32 were found to have an intact 7b gene whereas eight showed deletions of either three or 12 nucleotides. Surprisingly, among the eight viruses with deletions, three were from FIP diseased cats. These results show that deletions in the ORF7b gene are not constrained to low pathogenicity/enteric biotypes but also associated with pathogenicity/FIP biotypes of FCoV.

Clinicopathological findings and disease staging of feline infectious peritonitis: 51 cases from 2003 to 2009 in Taiwan

Fifty-one cats histopathologically confirmed to have been naturally infected by feline infectious peritonitis (FIP), were collected to analyse the clinical and laboratory findings and to characterise disease staging. Effusive FIP was found in 33 cats, non-effusive FIP in 12 cats, and mixed-type in six cats. Highly significant decreases in haematocrit and albumin levels and an increase in total bilirubin level were noted in both effusive and non-effusive FIP, at first presentation and before death. In serial blood examinations of the effusive group, anaemia and increases in bilirubin and aspartate aminotransferase (AST) were observed from 2 weeks to 0–3 days before death. The packed cell volume, bilirubin, AST, potassium, and sodium levels were established to predict disease staging and survival time. Cumulative points ranging from 0 to 4, 5 to 11 and excess of 12, indicate that the cat can survive for at least 2 weeks, less than 2 weeks and less than 3 days, respectively.

An outbreak of feline infectious peritonitis in a Taiwanese shelter: epidemiologic and molecular evidence for horizontal transmission of a novel type II feline coronavirus

Feline infectious peritonitis (FIP) is a fatal disease caused by feline coronavirus (FCoV) infection. FCoV can be divided into serotypes I and II. The virus that causes FIP (FIPV) is believed to occur sporadically and spread infrequently from cat to cat. Recently, an FIP outbreak from an animal shelter was confirmed in Taiwan. FCoV from all the cats in this shelter were analyzed to determine the epidemiology of this outbreak. Thirteen of 46 (28.2%) cats with typical signs of FIP were identified. Among them, seven cats were confirmed by necropsy and/or histopathological examinations. Despite the fact that more than one FCoV was identified in this multi-cat environment, the eight FIP cats were invariably found to be infected with a type II FCoV. Sequence analysis revealed that the type II FIPV detected from fecal samples, body effusions and granulomatous tissue homogenates from the cats that succumbed to FIP all harbored an identical recombination site in their S gene. Two of the cats that succumbed to FIP were found to harbor an identical nonsense mutation in the 3c gene. Fecal shedding of this type II virus in the effusive form of FIP can be detected up to six days before death. Taken together, our data demonstrate that horizontal transmission of FIPV is possible and that FIP cats can pose a potential risk to other cats living in the same environment.

The therapeutic efficacy of two antibabesial strategies against Babesia gibsoni.

Various combination strategies for treating Babesia gibsoni have been described. However, relapses after administering some combinations of antibabesial drugs and the presence of drug-resistant B. gibsoni still pose significant challenges to veterinarians. To compare the efficacy of a combination of clindamycin, diminazene, and imidocarb (CDI) to that of a combination of atovaquone and azithromycin (AA) for the treatment of B. gibsoni and to correlate drug efficacy with B. gibsoni mutations, 30 client-owned dogs with natural B. gibsoni infections were collected in the study. 17 dogs were treated with AA, and 13 dogs were treated with CDI combination. Hematological parameters were recorded on the day that the dogs were presented for treatment and during treatment. To detect the parasitic DNA, the B. gibsoni 18S rRNA gene was amplified, and to analyze the mutations, the cytochrome b (CYTb) gene was sequenced. The therapy duration for all of the dogs that recovered was 23.3±7.8 days in the AA group and 41.7±12.4 days in the CDI group. Nine of the 17 dogs in the AA group and 11 of the 13 dogs in the CDI group completely recovered. Seven dogs in the AA group and 2 dogs in the CDI group relapsed after treatment. The M121I mutation in the B. gibsoni CYTb gene was detected in all of the samples that were collected from AA-relapsed and AA-nonremission dogs. The dogs in the CDI group exhibited higher recovery rates and lower relapse rates during treatment for B. gibsoni infection. In addition, the detected M121I mutation was associated with AA treatment. The CDI combination is a promising alternative treatment strategy for B. gibsoni.

Influenza A(H6N1) Virus in Dogs, Taiwan.

We determined the prevalence of influenza A virus in dogs in Taiwan and isolated A/canine/Taiwan/E01/2014. Molecular analysis indicated that this isolate was closely related to influenza A(H6N1) viruses circulating in Taiwan and harbored the E627K substitution in the polymerase basic 2 protein, which indicated its ability to replicate in mammalian species.

Morphological and immunological evidence of a unique selective production and endoplasmic reticular accumulation of interleukin-1α in rat peritoneal macrophages induced by Pseudomonas aeruginosa exotoxin A

The immunotoxicity of Pseudomonas aeruginosa exotoxin A (ETA) on macrophages was evaluated by incubating rat peritoneal macrophages (RPM) with 1-100 ng/ml ETA for 3-60 h. Although the overall changes in cell viability and DNA, RNA, and protein synthesis of the ETA-treated RPM (E-RPM) were reduced in a dose- and time-dependent manner, there was a transient but evident rebound in RNA and/or protein synthesis at 24-36 h post-incubation (HPI) at 1-50 ng/ml ETA. However, a more apparent enhancement appeared in RNA and protein synthesis at 36-48 HPI in 10 and 50 ng/ml E-RPM after normalized on the basis of viable cell. Most 50-100 ng/ml E-RPM underwent necrosis/apoptosis before 24 HPI. By 36 HPI, 41% of 10 ng/ml E-RPM remained viable but were full of cytoplasmic granules due to the accumulation of glycoprotein in segmentally dilated endoplasmic reticulum. Immunological staining of the granules revealed strong IL-1alpha but weak or no signals for IL-1beta, IL-1 receptor antagonist, IL-6, and TNF-alpha. A time-dependent increase in IL-1alpha but no IL-1beta was detected in cell lysate of 10 ng/ml E-RPM; however, neither IL-1alpha nor IL-1beta was detected in culture supernatant. Thus, besides cytopathic and functional effects, ETA could induce a unique selective production and endoplasmic reticular accumulation of IL-1alpha in RPM.

A sensitive fluorescence in situ hybridization technique for detection of porcine reproductive and respiratory syndrome virus

A sensitive fluorescence in situ hybridization (ISH) for detecting porcine reproductive and respiratory syndrome virus (PRRSV) RNA in viral infected tissue was developed using digoxigenin-labeled RNA probes targeted on the nucleocapsid gene of PRRSV. In situ RNA/RNA hybrids were detected with an anti-digoxigenin antibody alkaline phosphatase conjugate and further revealed with Fast Red TR salt/naphthol AS-MX phosphate using a fluorescent microscope. Viral nucleic acid was readily demonstrated within macrophages, known to be the major target of PRRSV. In addition, positively stained cells were found in the salivary gland and skin tissues which have not been reported to contain PRRSV infected cells before. In conclusion, the fluorescence ISH used in this study provides a fast and sensitive means for screening virus-infected tissues in which relatively few cells are affected. This advantage will be especially beneficial for studying viral persistence and for routine diagnosis of PRRSV infection.

Dirofilaria immitis exposure status in client-owned cats with or without lower airway/lung-associated signs: case-control study in a canine heartworm-endemic area:

Objectives Heartworm-associated respiratory disease (HARD) is a recently recognised pathological manifestation in cats caused by Dirofilaria immitis exposure. This study aimed to estimate the percentage of cats at risk of developing HARD in a heartworm-endemic area (Taipei, Taiwan), and to test the correlation of heartworm exposure and the presence of lower airway/lung clinical signs (LA/L signs). Methods This was a prospective case-control study. The study design called for the enrolment of at least 80 cats with LA/L signs and at least 80 cats without such clinical signs in a 1 year period. The D immitis antibody seroprevalence of the two cohorts was compared. Results From February 2014 to January 2015, 187 client-owned cats were prospectively enrolled: 83 clinical cases with LA/L signs and 104 cats without such signs. Antibody seropositivity was approximately twice as frequent in cats with LA/L signs (13.3%) than in cats without signs (7.8%) (odds ratio [OR] 1.814); nevertheless, no statistically significant difference between the two cohorts (P = 0.22) was found. We used 41 frozen samples from free-roaming cats to examine the possibility of different exposure rates to mosquito bites between client-owned cats and stray cats, finding the seroprevalence to be 7.5% in free-roaming cats – a result not statistically different to that in client-owned cats (P = 0.60). Outdoor access was a significant risk factor for heartworm exposure in client-owned cats (OR 3.748; P = 0.03); however, living entirely indoors did not provide complete protection from exposure/infection. Conclusions and relevance Our results did not show statistically significant differences in antibody seroprevalence between cats with and without LA/L signs. LA/L signs were not always present under conditions of natural exposure. However, exposure to D immitis is not rare among client-owned cats, suggesting that heartworm prophylactics should be a part of routine care in all cats living in areas endemic for canine heartworm.

Detection of Porcine Reproductive and Respiratory Syndrome Virus by in situ Hybridization

A non-radioactive in situ hybridization technique based on the digoxigenin (DIG) labeling method was used for the detection of porcine reproductive and respiratory syndrome virus (PRRSV). Both sense and antisense DIG-labeled RNA probes targeted on the nucleocapsid gene of PRRSV were transcribed from a recombinant plasmid constructed from a local isolate (strain MD-001). A strong positive hybridization signal was obtained from a cell line, MARC-145, infected with PRRSV when the antisense RNA probe was used. In addition, the probe can detect cells infected with different strain of virus including US isolate (VR2332) and Lelystad virus without significant difference in signal intensity. Formalin-fixed paraffin-embedded tissues from the specific pathogen free pigs experimentally infected with PRRSV were examined using the same probe. Extensive to minimal positive signal was obtained from various organs examined. The cells showing positive hybridization signals were macrophages (in lymph nodes etc.) and Kupffer cells ( in liver) which were previously reported to be PRRSV permissive cells These results indicate that DIG-labeled RNA probe can be used routinely for the diagnosis of PRRSV infection.