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Volume 17 | October
Research Article | 04 Oct 2024
Prediction model for rectal temperature in cats with different baseline characteristics using a non-contact infrared thermometer
Nattakarn Naimon, Thitichai Jarudecha, Metita Sussadee, Rattana Muikaew, and Supochana Charoensin

Volume-17 | Issue-10 | Article-1 | https://doi.org/10.14202/vetworld.2024.2193-2203

Preview Abstract
Body temperature is the most useful clinical parameter for evaluating animal health. In clinical practice, rectal temperature is the gold standard for assessing body temperature, but rectal temperature measurement is not convenient and can cause stress in animals. The non-contact infrared thermometer is considered an alternative method for skin temperature measurements in animals. Many biological factors may influence the response of body regions to thermal challenges; thus, the identification of these variables is essential for accurate infrared temperature measurements. This study aimed to estimate the relationship between the physiological factors of cats and their body temperature measured across various body positions, as well as to propose a model for predicting rectal temperature using an infrared thermometer.  A total of 184 client-owned cats were included in this study. The infrared temperature (<sup>°</sup>F) was measured using a non-contact infrared thermometer at five body positions: maxillary canine gingival margin (GCT), anal skin (ANS), inguinal canal (ING), ear canal (EC), and palmar pad. The five biological factors (age, body condition score [BCS], gender, hair type, and hair color) were recorded and analyzed to adjust predictive factors for rectal temperature prediction. All statistical analyses were performed using multivariable linear regression. The rectal temperature prediction model was then designed using the forward stepwise selection method.  Based on multivariable linear regression analysis of infrared temperature results, the pre-prediction model showed significant correlations with rectal temperature for ANS, GCT, and EC (p = 0.0074, 0.0042, and 0.0118, respectively). Moreover, the combination of infrared temperatures on ANS and ING was the most appropriate parameter for predicting rectal temperature (p = 0.0008). All models were adjusted according to the baseline characteristics of the cats. However, the adjusted R-squared values of the pre-prediction model of the infrared temperature on the ANS, GCT, and EC and the final prediction model by the infrared temperature on the ANS combined with the ING were low (8.7%, 8.9%, 7.3%, and 12.8%, respectively).  The prediction model of rectal temperature of cats by infrared temperature from a non-contact infrared thermometer in ANS combined with ING and adjusted by age, BCS, hair type, and hair color may be applicable for use in clinical practice. This study found that the adjusted R-squared values of all models were low; the predictive model will need to be developed and used to test validity and reliability with an external study group for assessing their practical usefulness. 
Research Article | 04 Oct 2024
Evaluation of the effects of green tea extract as a dietary supplement in sheep on gas production, volatile fatty acids, and digestibility
Ririn Siti Rahmatillah, Diky Ramdani, Iman Hernaman, Anuraga Jayanegara, and Nanik Hidayatik

Volume-17 | Issue-10 | Article-2 | https://doi.org/10.14202/vetworld.2024.2204-2210

Preview Abstract
Phenolic tannins, which are ubiquitous in plants, exhibit diverse biological activities and have drawn significant attention for their potential impact on ruminant nutrition and health. Although phenolic tannins have beneficial and detrimental effects on rumen fermentation, their precise influence remains poorly understood. This study aimed to explore the effects of varying doses of green tea extract (GTE) on rumen fermentation parameters through an in vitro trial using sheep rumen liquids.  A 4 × 2 factorial design was used to test the effect of 4 different doses of GTE treatment (0, 140, 280, and 560 mg/kg) in 2 different in vitro runs on degradability, fermentation profiles, and gas production using the in vitro Hohenheim Gas Test method.  Across running times, the GTE-treated diet did not affect (p > 0.05) dry matter degradability % and organic matter degradability %, pH, ammonia (NH3-N, mg/dL), 24 h total gas production (tGP 24h, mL), and acetate-to-propionate ratio (A: P), but it reduced (p < 0.05) tGP 6 h compared with GTE-0 (control diet without GTE). GTE treatment tended to reduce (p < 0.1) methane (CH4, % LEL) and total volatile Fatty Acids (tVFA, mM). Across GTE treatment, the 1st in vitro run had higher (p < 0.001) tGP 6–24 h and pH, but lower (p < 0.001) tVFA and A: P in comparison with the 2<sup>nd</sup> in vitro run.  GTE treatment tends to decrease CH4 output in rumen without affecting degradability, tGP, and most fermentation profiles, except for a tendency to reduce tVFA. 
Research Article | 04 Oct 2024
Therapeutic potential of green-synthesized silver nanoparticles: Combating biofilms of multidrug-resistant Staphylococcus aureus RM-Ph8 and modulating the immune response in the liver tissue of rats
Mohamed T. Shaaban, Sahar H. Orabi, Marwa Salah Abdel‑Hamid, Reda M. S. Korany, Fatimah M. Alshehrei, and Rania Hamed Elbawab

Volume-17 | Issue-10 | Article-3 | https://doi.org/10.14202/vetworld.2024.2211-2224

Preview Abstract
The emergence of multidrug-resistant Staphylococcus aureus (MRSA) strains poses a significant threat to healthcare settings. Although various studies have explored alternative antibiotics, discovering novel therapeutic agents remains crucial. This study aimed to synthesize green silver nanoparticles (AgNPs) as bactericidal agents, identify a multidrug-resistant isolate of Staphylococcus aureus, and explore their biofilm formation ability. To estimate the role of phyto-AgNPs in the perfection of immune markers and healing hepatic lesions in vivo.  The clinical isolate of MRSA was identified using 16S rRNA New green AgNPs derived from Artemisia annua extract were synthesized. The nanoparticles (NPs) were characterized, and their minimum inhibitory concentration was estimated for fighting MRSA biofilm. A study was conducted on rats to evaluate the effect of new NPs on their immune response to MRSA infection.  The new clinical isolate of MRSA RM-Ph8 was identified by molecular phylogenetic analysis as S. aureus, and 16S rRNA sequence analysis confirmed that the new strain was similar to S. aureus with 98.12% identity with accession number OQ421819. The FTIR of the new phyto-AgNPs displayed different functional groups that work as reducing silver nitrate agents. Transmission electron microscopy and scanning electron microscopy images showed spherical particles with an average diameter of 6–28 nm smaller. The chemical method led to complete cell destruction of the multidrug strain within 24 h. Biofilm formation showed that the new MRSA clinical strain was strongly adherent (88%). Notably, the phyto-AgNPs exhibited significant bactericidal activity against the new MRSA strain, with an MIC of up to 50 mg/mL. Moreover, phyto- AgNPs significantly decreased reversed MRSA-induced liver and kidney function impairment, with improvement in both the histopathological lesions and immune histochemical expression of tumor necrosis factor-α and inducible nitric oxide synthase at p < 0.05 compared with the untreated group.  Green AgNPs are a promising therapeutic approach against multidrug-resistant bacterial infections, surpassing the effectiveness of conventional antibiotics. 
Research Article | 04 Oct 2024
Histopathological patterns and immunophenotyping of feline lymphomas and incidence in Metropolitan Bangkok, Thailand
Jedsada Siripoonsub, Sirintra Sirivisoot, Somporn Techangamsuwan, and Anudep Rungsipipat

Volume-17 | Issue-10 | Article-4 | https://doi.org/10.14202/vetworld.2024.2225-2234

Preview Abstract
Feline lymphomas are categorized based on the location of tumor cells, with anatomical classifications including alimentary, mediastinal, multicentric, and extranodal forms. Accurate diagnosis and classification of feline lymphoma are paramount for enhancing treatment and prognosis. T-cell lymphomas are CD3 positive, while B-cell lymphomas exhibit positive for CD20, CD79α, and paired box 5 (PAX5). The aims of this study were (1) to classify feline lymphoma in each anatomical subtype using the World Health Organization (WHO) classification to provide information on epidemiological findings; (2) to investigate the expression and detection of B-cell lymphoma, various antibodies will be used, with the addition of PAX5, for clearer results; and (3) to gather more extensive information about feline lymphoma in Thailand, particularly in the Bangkok area.  From 2011 to 2023, 86 sample tissues were submitted for routine pathological examination at the Department of Pathology, Faculty of Veterinary Science, Chulalongkorn University. Immunohistochemistry (IHC) was performed to detect an immunophenotype of PAX5, CD79α, CD20 (B-cell lineage), and CD3 (T-cell lineage). Eighty-six formalin-fixed, paraffin-embedded lymphoma tissues were prepared on silane-coated slides. After IHC, all cases were classified according to the WHO classification.  The most common form of lymphoma in this study was extranodal lymphoma at 37.2% (32/86), followed by multicentric lymphoma at 31.3% (27/74), mediastinal lymphoma at 17.4% (15/86), and alimentary lymphoma at 14% (12/86). Most extranodal lymphoma cases were in the nasal region. From the anatomical form, multicentric and extranodal lymphomas were predominantly diffuse large B-cell high-grade, while mediastinal lymphomas were small low-grade B-cell lymphomas. Alimentary lymphomas occur in various types, with most being the B-cell type.  This study indicates that extranodal lymphoma and extranodal lymphoma are the most frequent presentations found in cats in Bangkok. Mediastinal and alimentary lymphomas still occur. The utilization of various B-cell markers in combination could aid pathologists in distinguishing between various stages of B-cell maturation, assessing tumor cell heterogeneity, and determining the phenotype in scenarios where there is a loss of common B-cell markers diffuse large B-cell lymphomas is the most prevalent subtype of feline lymphoma. Significantly, relying solely on immunochemistry with one parameter may not be sufficient for a definitive diagnosis of B-cell lymphoma, as another parameter may also be necessary. 
Research Article | 07 Oct 2024
Designing a multi-epitope construct using immuno-informatic tools to prepare a messenger RNA vaccine against Rhipicephalus microplus ticks
Ubaid Ullah, Kamran Ashraf, Wasim Shehzad, and Muhammad Imran Rashid

Volume-17 | Issue-10 | Article-5 | https://doi.org/10.14202/vetworld.2024.2235-2247

Preview Abstract
Ticks are blood-feeding ectoparasites that transmit pathogens to animals and humans. One of the most important hard ticks in animals is Rhipicephalus microplus, which transmits Babesia and Anaplasma spp. Although many potential tick vaccine candidates have been identified, no effective vaccine that can provide sterile immunity against R. microplus tick infestations has been developed. This study aimed to design a construct using different computational tools to identify and predict immunogenic epitopes within protein sequences and to prepare a messenger RNA (mRNA) vaccine against R. microplus based on lipid nanoparticles (LNPs).  The R. microplus proteins (Bm86, Subolesin, and ATAQ) were selected and their consensus sequence was obtained from the National Center for Biotechnology Information in FASTA format. The Immune Epitope Database and Analysis Resource (IEBD) server was used for the prediction of helper T-cell epitopes, the NetCTL 1.2 server was used to predict cytotoxic T-cell epitopes, and the ABCpred server was used for B-cell epitope prediction. Antigenicity testing, allergenicity assessment, and toxicity screening were immuno-informatic techniques used to identify potent epitopes within protein sequences. The multi-epitope construct was prepared and cloned into the pVAX1 plasmid. Plasmids were transformed in compatible competent cells, and restriction analysis was performed. After restriction analysis of the transformed plasmid, in vitro transcription was performed to prepare mRNA. The mRNA was purified, quantified, and converted into complementary DNA, and gene-specific primers were used to confirm the in vitro transcription of mRNA. A mixture of four lipids containing 1,2-dioleoyl-3-dimethylammonium-propane (DODAP), Distearoylphosphatidylcholine (DSPC, cholesterol, and 1,2-Dimyristoyl-sn-glycero-3-methoxypolyethylene glycol-2000 (DMG PEG-2000 was used to prepare LNPs. LNPs were characterized using a scanning electron microscope, Zeta potential, and Zeta Sizer tests.  More than 1000 epitopes were predicted, from which only nine helper T-lymphocytes, 18 cytotoxic T-lymphocytes, and nine B-cell epitopes of all three proteins were selected with high antigenic scores of 0.958 for Bm86, 0.752 for Subolesin, and 0.964 for ATAQ, respectively. An adjuvant was used to enhance immune responses, all of which were linked to one another using GPGPG, AAY, and KK linkers, respectively. The physiochemical properties predicted that the instability index of the construct would be <40%, indicating that the construct is stable. Plasmids were transformed in compatible competent cells, and white-transformed colonies were observed. Restriction analysis was performed, DNA was transcribed into mRNA, and LNPs were prepared and characterized.  More than 1000 epitopes were predicted using immune informatic tools, and only high-scoring epitopes were selected. A multi-epitope construct was designed using bio-informatic tools, and its physicochemical properties were predicted. The design construct was inserted into the pVAX1 plasmid, and in vitro transcription was performed to prepare the mRNA. LNPs of mRNA were prepared and characterized to be used as vaccines. It was found that LNPs were stable and nanometer-sized. 
Research Article | 07 Oct 2024
Scanning of antennae and maxillary palps of anthropophilic Aedes aegypti and ornithophilic Culex pipiens as potential arbovirus vectors
Faten Abouelmagd, Faten Abouelmagd, Mohamed Elmutasim Elsheikh, Elshiekh Khidir, Mohammed Radwan, Karim Mohamed Rashad, and Manal El Said, and Manal El Said

Volume-17 | Issue-10 | Article-6 | https://doi.org/10.14202/vetworld.2024.2248-2252

Preview Abstract
Efficient mosquito vectors are required to persist and propagate arthropod-borne diseases that seriously affect impoverished populations worldwide. Mosquito sensilla plays a crucial role in host-seeking and disease transmission to humans. This study aimed to distinguish between the several types of sensilla found on the antennae and maxillary palps of Culex pipiens and Aedes aegypti, matching this diversity with host preference and disease transmission.  Overall, 1300 mosquitoes were collected and examined using dissection and light microscopy. Scanning electron microscopy was used to identify and describe the diverse types of sensilla found on the antennae and maxillary palps of C. pipiens and A. aegypti.  In total, 900 C. pipiens and 400 A. aegypti mosquitoes were identified. The antennae and maxillary palps of C. pipiens and A. aegypti carry both sensilla trichoidea and sensilla chaetica. The C. pipiens antenna has long and short grooved peg sensilla, whereas A. aegypti lacks long pegs and expresses only occasional short pegs. The maxillary palps express Capitate pegs in both mosquito species and exclusively show sensilla campaniform in A. aegypti.  The lack of long-grooved pegs and the presence of few short pegs, along with campaniform sensilla, limit the host range of A. aegypti and reduce its susceptibility to many infections, unlike C. pipiens. 
Review Article | 07 Oct 2024
Low-dose radiation research insights in experimental animals: A gateway to therapeutic implications
Nayanatara Arunkumar, Megha Gokul, Harini Narayanam, and A. K. Ananya

Volume-17 | Issue-10 | Article-7 | https://doi.org/10.14202/vetworld.2024.2253-2258

Preview Abstract
In recent years, a significant research effort has been underway to explore the effects of low-dose radiation (LDR). Animal models play a key role in various fields of research, including biomedicine, pharmaceutical, environmental, and behavioral studies. The use of animal models has been an invaluable tool in radiation research for understanding radiation biology, assessing radiation risks, and developing strategies for radiation protection and medical management. In the present review, the initial part focuses on the deleterious effects of high-dose radiation, and in correlation to that, in the later part of the review, the emphasis has been given to experimental approaches to explore the beneficial effects of LDR using animal models. This review could help explore the innovative approach for future research targeting the therapeutic role of LDR in various diseases, including depression, Cancer, Parkinson’s disease, and Alzheimer’s disease. 
Research Article | 07 Oct 2024
First detection and impact of bovine herpesvirus type 4 on dairy cattle reproduction in Thailand
Ruchikon Jongsuwanwattana, Ruchikon Jongsuwanwattana, Piyathip Setthawong, Siriwat Suadsong, Sudson Sirivaidyapong, and Theerawat Swangchan-Uthai, and Theerawat Swangchan-Uthai

Volume-17 | Issue-10 | Article-8 | https://doi.org/10.14202/vetworld.2024.2259-2266

Preview Abstract
Postpartum reproductive tract infections pose significant challenges to dairy farms, leading to economic losses due to reduced fertility associated with uterine inflammation. In veterinary practice, numerous research groups have explored the underlying causes of subfertility in cows, including surveying endemic viral infections related to endometritis in local areas. This study investigated bovine herpesvirus 4 (BoHV-4) infection in Thai dairy herds and assessed its impact on endometritis and subsequent reproductive outcomes.  The present study analyzed BoHV-4 DNA in various samples, including milk, blood, and endometrial tissue, from 44 Holstein-Friesian cows 21–47 days postpartum across five dairy herds in Central Thailand. BoHV-4 glycoprotein B and thymidine kinase DNA sequences were detected using the polymerase chain reaction (PCR) and nested PCR, with sequence comparisons made to GenBank data for phylogenetic analysis. The endometritis status was diagnosed through vaginal mucus examination and endometrial cytology, with reproductive performance monitored up to the subsequent calving.  BoHV-4 DNA was identified in blood and endometrial tissues (15.91%) but not in milk samples. Phylogenetic analysis revealed that the local BoHV-4 strains are similar to those identified in Brazil and Japan. Notably, the presence of BoHV-4 was correlated with reduced postpartum reproductive performance, particularly extending the interval from calving to the first service.  Our findings underscore the importance of integrating BoHV-4 genomic surveys and uterine health assessments to refine reproductive management strategies within the dairy industry. 
Review Article | 07 Oct 2024
Fish diversity assessment through conventional morphological identification and recent advances in Saudi Arabia: A review
Muhammad Browijoyo Santanumurti, Muhammad Browijoyo Santanumurti, Muhammad Ar Rozzaaq Nugraha, Novi Rosmala Dewi, Muhammad Awaluddin, Pei Wen Tang, Helen Indah Pardede, Lafi Al Solami, Laksmi Sulmartiwi, and Mohamed Ahmed Abu El-Regal, and Mohamed Ahmed Abu El-Regal

Volume-17 | Issue-10 | Article-9 | https://doi.org/10.14202/vetworld.2024.2267-2285

Preview Abstract
Fish identification in the Red Sea, particularly in Saudi Arabia, has a long history. Because of the vast fish diversity in Saudi Arabia, proper species identification is required. Indeed, identifying fish species is critical for biodiversity conservation, food and drug safety, and sustainable fishery management. Numerous approaches have been used to identify fish species, including conventional morphological identification, next-generation sequencing (NGS), nanopore sequencing, DNA barcoding, and environmental DNA analysis. In this review, we collected as much scientific information as possible on species identification in Saudi Arabia. Our findings suggest that the identification process has advanced and spread rapidly and broadly, as evidenced by the discovery of new fish species in Saudi Arabia. The advantages and disadvantages of each method were discussed as part of a comprehensive comparison. This study aimed to provide further scientific knowledge to promote the growth of fish diversity worldwide. 
Research Article | 17 Oct 2024
Molecular detection and quantification of canine parvovirus 2 using a fast and sensitive SYBR® green-based quantitative polymerase chain reaction assay in dogs affected with gastroenteritis
Anthony Loor-Giler, Anthony Loor-Giler, Sara Castillo-Reyes, Silvana Santander-Parra, Martín Campos, Martín Campos, Renán Mena-Pérez, Renán Mena-Pérez, Santiago Prado-Chiriboga, Santiago Prado-Chiriboga, and Luis Nuñez, and Luis Nuñez

Volume-17 | Issue-10 | Article-10 | https://doi.org/10.14202/vetworld.2024.2286-2294

Preview Abstract
Viral gastroenteritis in canines is primarily caused by the canine parvovirus 2 (CPV-2). Infections by this virus can cause severe consequences in dogs, such as fever, vomiting, diarrhea, septicemia, systemic inflammation, and immunosuppression. Therefore, the mortality rate of persistent infections caused by this virus is significantly high. The capsid protein VP2 genome of canine parvovirus has undergone many changes, resulting in the emergence of different genotypes, including CPV-2a, CPV-2b, and CPV-2c. Diagnostic procedures often lack the necessary specificity for early infection diagnosis. Early detection of the infection enhances the likelihood of canine survival because the canine will receive prompt therapy. Hence, this study aimed to develop a quantitative polymerase chain reaction (qPCR)-based diagnostic technique using SYBR Green for the rapid and accurate detection and quantification of CPV-2.  The assay was specifically designed to identify a portion of the conserved NS gene using primers that amplify a 125-bp fragment. The qPCR method was executed in the fast mode to expedite the process using Power up SYBR Green Master Mix reagent. A standard curve was constructed using the amplified and purified PCR product of the NS gene.  The limit of detection and quantification were determined in the one amplified-DNA copy. The standard curve showed an efficiency of 99.5% and inter- and intra-assay coefficients of variation of 0.387%–0.976% and 0.085%–0.430%, respectively. The assay was specific for the amplification of CPV-2, as no amplification was observed for other viral genomes (canine adenovirus II, canine distemper virus, canine coronavirus, and canine astrovirus) or from the negative controls. Inter- and intra-tests for repeatability showed low test variability around the run time. To validate the present assay, 200 samples of fezzes from canines with gastroenteritis and symptoms associated with enteric infection were tested using the qPCR protocol. From the analyzed samples, 136 were positive for CPV-2 by qPCR assay, of which 110 were before diagnostic positive for the virus by endpoint PCR, showing high sensitivity of the current assay. CPV-2 was detected in dogs over 2 weeks old up to dogs 9 years old, where the highest viral concentration found was 16429595 gene copies in dogs aged 2 weeks.  In the present study, a rapid, specific, repeatable, and sensitive assay was developed for the detection and quantification of CPV-2. Furthermore, it was demonstrated that in the population of domestic dogs in Ecuador affected with gastrointestinal disease, the virus is presented in dogs of different ages and not only in young dogs. 
Research Article | 17 Oct 2024
Genetic diversity and polymorphism of genes associated with meat quality and growth traits of A Luoi Yellow cattle in Vietnam
Thu Nu Anh Le, Huong Thi Duong, Phuong Thi Lan Le, Thuong Thi Nguyen, Thuan Thi Duy Phan, Phung Dinh Le, and Van Huu Nguyen

Volume-17 | Issue-10 | Article-11 | https://doi.org/10.14202/vetworld.2024.2295-2303

Preview Abstract
A Luoi Yellow cattle is an indigenous cattle breed that is raised in the A Luoi District, Vietnam, characterized by its small body size, high adaptability, and meat quality favored by domestic consumers. Marker-assisted selection is an effective approach for improving breeding genetics and sustainably developing livestock production. Therefore, this study aimed to investigate the genetic diversity and polymorphism of genes associated with meat quality and productivity in the A Luoi Yellow cattle population with the goal of future breeding selection and sustainable development of the A Luoi Yellow beef brand.  In this study, we genotyped six functional genes, including Leptin (LEP), Calpastatin (CAST), Calpain 1, pleomorphic adenoma gene 1, Sirtuin 1, and Sirtuin 2 (SIRT2), involved in meat quality and growth traits using polymerase chain reaction (PCR) or PCR-restriction fragment length polymorphis. We also investigated mitochondrial DNA (mtDNA) and the Y chromosome-specific gene on the Y chromosome to elucidate the genetic diversity and paternal and maternal origin of the A Luoi Yellow cattle using Sanger sequencing.  The results showed that A Luoi yellow cattle have Bos indicus origin from both paternal and maternal lineages. By mtDNA analysis, we identified two new haplotypes of the I1 haplogroup that were not previously detected. The genotyping of the six functional genes indicated that A Luoi Yellow cattle carry favorable alleles that increase meat tenderness and body size, with frequencies of 0.02–0.40. In particular, the presence of desirable homozygous genotypes of the CAST, LEP, and SIRT2 genes will be important for the future selection of animals based on their potential performance in meat quality and productivity.  The findings of this study is useful for the future breeding and sustainable development of A Luoi Yellow cattle. 
Research Article | 17 Oct 2024
Exonic mutations of POU class 1 homeobox 1 are associated with milk pH in high-producing Holstein Friesian cows
Muhammad Cahyadi, Ammar Ibnu Hasan, Djorodjatun Samodro Sakti, Nasta ‘Ainun Nissa, Ahmad Pramono, Suryo Firmanto, Rizwan Nur Friyatna, Slamet Diah Volkandari, and Pita Sudrajad

Volume-17 | Issue-10 | Article-12 | https://doi.org/10.14202/vetworld.2024.2304-2310

Preview Abstract
Milk physicochemical properties play essential role in the milk processing industry, which are moderately to highly affected by genetic factors. This study aimed to evaluate the association between single nucleotide polymorphisms in POU class 1 homeobox 1 (POU1F1) and the physicochemical properties of milk in high-producing Holstein Friesian (HF) cows.  A total of 149 high-producing dairy cows from PT Ultra Peternakan Bandung Selatan was included in this study. The physicochemical properties of milk, including density, freezing point, pH, lactose, solid non-fat, protein, and ash content, were determined. Moreover, three polymorphisms within the exon regions of POU1F1 (c.195G>A, c.300G>T, and c.828G>A) were analyzed using polymerase chain reaction-restriction fragment length polymorphism. The association between these polymorphisms and the physicochemical properties of milk was determined using a mixed-effects model analysis, in which the lactation period was used as a covariate.  This study found that two polymorphisms, c.195G>A and c.828G>A, significantly affected the pH of fresh milk. Cows with both the GG genotypes c.195G>A and c.828G>A had lower milk pH values than those with the other genotypes. In addition, a non-significant effect of POU1F1 was observed on the other physicochemical properties of milk.  Two POU1F1 polymorphisms determined the pH of fresh milk in the Indonesian HF population. These are potential marker candidates for milk pH that directly affect the development of dairy products in the milk processing industry. 
Research Article | 17 Oct 2024
Beyond traditional vaccines: Semi-purified low-pathogenic avian influenza H9N2 virus-like particles and their promise for broiler immunity
Muhammad Luqman, Sajjad Ur Rahman, Shafia Tehseen Gul, and Muhammad Shahid Mahmood

Volume-17 | Issue-10 | Article-13 | https://doi.org/10.14202/vetworld.2024.2311-2321

Preview Abstract
Avian influenza is a global threat to avian species, particularly in developing countries. Recombinant vaccines, including virus-like particles (VLPs), are promising strategies for preventing the spread of the disease. VLPs produced through the self-assembly of viral structural proteins without genomic material mimic native virions and are promising platforms for new vaccines. VLPs have been shown to elicit protective antibodies and are effective and safe vaccines against influenza. This study aimed to optimize the protocol for the production and characterization of H9N2 VLPs and their evaluation as a vaccine in broiler birds.  Low-pathogenic influenza virus (LPAI) H9N2 was isolated and characterized through whole-genome sequencing, and a VLP-based vaccine for LPAI H9N2 was prepared using a baculovirus expression system. Codon-optimized hemagglutinin (HA), neuraminidase (NA), and M1 were successfully cloned in pFastbac1 and expressed in SF9 cells. Proteins were characterized using sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE), western blotting, and electron microscopy after purification. Semi-purified proteins were tested as a vaccine in broiler chickens challenged with LPAI H9N2.  Recombinant Bacmid DNA from positive clones was extracted and confirmed using a polymerase chain reaction. The transfection showed cytopathic effects, and the proteins were confirmed through western blotting and SDS-PAGE, which showed the sizes of HA = 62–64 KD, NA = 52 KD, and M1 = 25 KD. The shape and morphology were confirmed through transmission electron microscopy which revealed 100–150 nm size particles. As a result, the semi-purified VLPs (HA assay: 256) were tested as a vaccine for specific-pathogen free broiler birds; administered through subcutaneous and intranasal routes. The birds were challenged on the 28th day after vaccination with the H9N2 strain, and the birds showed significant cross-reactivity with the H9N2 strain.  The semi-purified VLP-based vaccine induced a significant immune response in vivo. This vaccine formulation has the potential to control avian influenza outbreaks in Pakistan’s poultry industry. 
Research Article | 17 Oct 2024
Optimizing the incubation conditions of third-stage larvae of the camel nasal bot Cephalopina titillator (Diptera: Oestridae) for harvesting adult flies
Mohammad Nafi Solaiman Al-Sabi, Hams Almohammed, Fatema Alghatam, Ghadeer Alhafiz, Omar Al-Jabr, and Ahmed M. A. Meligy, and Ahmed M. A. Meligy

Volume-17 | Issue-10 | Article-14 | https://doi.org/10.14202/vetworld.2024.2322-2328

Preview Abstract
Understanding the developmental conditions of Cephalopina titillator larvae and their effect on the success of pupation and adult emergence can help prevent and control this disease in camels. Incubating C. titillator larvae in vitro requires optimized conditions that have not been adequately reported in the literature. This study aimed to optimize conditions for harvesting adult flies from third-stage larvae (L3).  L3 collected from naturally infested camels was washed in sterile saline, weighed, and placed in vials containing local sand. The vials were covered with gauze and incubated at 30<sup>°</sup>C–36<sup>°</sup>C with 60%–62% relative humidity in an environmental chamber.  A minimum critical weight of 754 mg per larva was found to be essential for the successful eclosion of the adults, regardless of the sex of the emerged flies. The pupariation period lasted 1–8 days (d) and took 5–13 days. Most incubated L3 formed puparia, but most failed to emerge as adults. The success rate of eclosion was 37.1%. The hatched adults survived for up to 18 days, and the females survived longer (12 d; 6–18) than the males (8.1 d; 3–16).  The higher eclosion success tendency of certain sand types might be influenced by the sand’s physical and/ or chemical characteristics. The current in vitro conditions resemble those during the hot seasons and are suitable for harvesting viable adults of C. titillator from L3. 

Volume-17 | Issue-10 | Article-15 | https://doi.org/10.14202/vetworld.2024.2329-2337

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The use of food dyes can cause certain diseases, such as anemia and indigestion, along with other disorders, tumors, and even cancer. Therefore, this study aimed to determine the chemical nature and toxicity of some commercial dyes locally used in processed foods compared with standard food dyes.  Three types of standard and commercial food color additives (Sunset Yellow, Tartrazine, and Carmoisine) were extensively examined. The chemical structures and functional groups of the dyes were evaluated by Fourier-transform infrared (FTIR) spectroscopy. The melting temperatures of the dyes were also determined by chemical thermal analysis. The acute toxicity test to evaluate the standard and commercial food color safety was estimated by a range-finding study using 150 Wistar albino rats. Sub-groups were administered one of the three colors under study at doses of 2, 3, 4, and 5 g/kg body weight (BW) orally for 7 days. When no mortality was observed, an additional 15 g/kg BW was administered. Concerning the median lethal dose 50 (LD<sub>50</sub>), 38 rats were exploited using the up-and-down method.  Commercial dyes had lower melting points than standard colors. Regarding the range-finding study, rats receiving different doses of the dyes exhibited no signs of toxicity, no deaths, and no clinical or gross pathological signs throughout the 7 days of the experiment. However, the animals that were dosed with 15 g/kg BW of each dye showed signs of loss of appetite, tachycardia, drowsiness, and eventual death. The LD<sub>50</sub> values of the commercial food dyes, particularly Sunset Yellow and Carmoisine, were lower than those of the standard dyes.  Commercial food colors were more toxic to rats than standard food colors. Differences were observed between the purity of the standard and commercial dyes, and the latter ones contained different percentages of salt, indicating the occurrence of fraud in commercial markets. 

Volume-17 | Issue-10 | Article-16 | https://doi.org/10.14202/vetworld.2024.2338-2346

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Antimicrobial resistance in poultry farms is a significant global public health concern that has led farmers to explore alternative antibiotics, such as prebiotics in poultry production. This study aimed to examine the antimicrobial activity of ethanolic shallot extract (ESE) and the effects of adding shallot powder (SP) to broiler feed on broiler growth, immune response to Newcastle disease virus (NDV) vaccination, and gastrointestinal tract bacteria.  We determined the antimicrobial effects of ESE against Escherichia coli O157:H7 (EOH) and Lactobacillus acidophilus TISTR 2365 (L2365) using the agar well diffusion method. We used a complete randomized design to assign 120 1-day-old Arbor Acre chicks to six groups with four replicates of five broiler chickens over 42 days. The treatment groups were as follows: T1-basal diet (B) + NDV vaccination (positive control), T2-B (negative control), T3-B + 2 g SP per kg of feed, T4-B + 2 g SP per kg of feed + NDV, T5-B + 4 g SP per kg of feed, and T6-B + 4 g SP per kg of feed + NDV.  The minimum inhibitory concentrations of ESE on EOH and L2365 were 62.50 and 125.00 mg/mL, respectively. The body weight gain, average daily gain, and feed conversion ratio in the 4 g SP of T5 and T6 groups were significantly improved compared with the other groups (p < 0.05). The immune organ (IO) and thymus gland weights in the T4 group were significantly greater than those observed in the positive and negative control groups (p < 0.05). The IO weights of the bursae of Fabricius and spleen tended to be greater in the T4 group than in the other groups. T5 group broilers had the highest ratio of villus height to crypt depth. The humoral immunity titers against NDV vaccination were improved in the SP-supplemented groups compared with the non-supplemented groups (p > 0.05). SP supplementation reduced the levels of coliform (p < 0.05) and E. coli in the broiler intestine by adding 4 g of SP per kg of feed. However, L2365 was more tolerant to ESE in vitro and tended to increase in line with increased SP levels.  ESE showed strong antimicrobial activity to reduce harmful bacteria, and SP supplementation may exhibit prebiotic effects to increase broiler chicken growth, immunity, and microbial balance. 
Research Article | 27 Oct 2024
Cross-sectional study of antibiotic resistance to Escherichia coli and Salmonella spp. in cats in Yogyakarta, Indonesia, and Dili, Timor-Leste
Widagdo Sri Nugroho, Antonino Do Karmo, Gustaf Eifel Silalahi, Elphan Augusta Kajang, and Putu Cri Devischa Gallantiswara

Volume-17 | Issue-10 | Article-17 | https://doi.org/10.14202/vetworld.2024.2347-2354

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Antibiotics are used in veterinary clinics and animal hospitals to treat infectious diseases. However, the improper use of antibiotics causes antibiotic resistance, which threatens future disease therapeutics in pet animals. This study aimed to estimate the prevalence of Escherichia coli and Salmonella spp. in cats and their resistance to antibiotics in Yogyakarta Province, Indonesia (IDN), and Dili, Timor-Leste (TL).  A total of 255 cat’s rectal swab samples from veterinary clinics and hospitals in Yogyakarta Province, IDN, and Dili, TL were collected. All samples were transferred aseptically into an enrichment medium and subjected to various culture tests for E. coli and Salmonella spp. identification. All identified isolates were tested for antibiotic sensitivity using Kirby−Bauer disk diffusion method.  This study successfully isolated E. coli from 172/255 (67.45%) rectal swab samples, that is, 122/188 samples (64.89%) from Yogyakarta Province, IDN, and 50/67 samples (74.6%) from Dili, TL. Salmonella spp. was isolated from 13/188 samples (6.91%) from Yogyakarta, IDN. The antibiotic susceptibility test indicated that more than 30% of E. coli were resistant to ampicillin (AMP) (IDN = 39.3%, TL = 50%) and tetracycline (TE) (IDN = 41.8%, TL = 42%), and more than 40% of Salmonella spp. were resistant to enrofloxacin (44%), TE (56%), streptomycin (61%), and AMP (83%).  E. coli and Salmonella spp. succeeded isolation in cats from IDN and TL, and some isolates were resistant to antibiotics. Cats with diarrhea or digestive problems have a 9.5-fold increased risk of infection by Salmonella spp. Considering the prevalence of resistance to E. coli and Salmonella spp., it is important to manage antibiotic resistance distribution across companion animals and humans because both species share the same living environment. 
Research Article | 27 Oct 2024
Analysis of species adulteration in beef sausage using real-time polymerase chain reaction in Makassar, Indonesia
Mirna Mualim, Mirna Mualim, Hadri Latif, Herwin Pisestyani, and Puji Rahayu

Volume-17 | Issue-10 | Article-18 | https://doi.org/10.14202/vetworld.2024.2355-2364

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Adulteration, or the inclusion of meats not declared on the label of processed meat products, constitutes a fraudulent practice that poses a threat to public health. Sausages, which are processed meats derived from a blend of minced meats that obscure the original muscle’s morphological features, are particularly prone to adulteration, making the visual detection of fraud more challenging. The research aimed to detect and measure the proportion of pork, chicken, buffalo, and beef DNA in commercially available processed meat packaged, labeled, and sold as “beef sausages” in Makassar, Indonesia.  A total of 30 beef sausage samples were collected from traditional and modern markets as well as tourist attractions in Makassar. DNA was isolated and the species were identified using quantitative polymerase chain reaction.  The findings revealed that all sausage samples contained not only beef DNA, as indicated on their labels but also undeclared DNA from chicken and buffalo. Notably, pork DNA was not detected in the samples. The frequencies of chicken and buffalo meat were 9.2% and 10%, respectively, whereas it was 0.85% for beef in the beef sausage samples.  The discovery of chicken and buffalo species in beef sausage samples indicates adulteration, potentially posing severe quality risks. 
Research Article | 27 Oct 2024
Testosterone hormone levels and breast muscle performance of Pelung chickens after zinc sulfate and synthetic testosterone supplementation
Rizki Fitrawan Yuneldi, Claude Mona Airin, Hendry T. S. Saragih, Andhika Yudha Prawira, and Pudji Astuti

Volume-17 | Issue-10 | Article-19 | https://doi.org/10.14202/vetworld.2024.2365-2369

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Pelung chicken Gallus gallus gallus (Linnaeus, 1758) is a chicken endemic to Cianjur, West Java, Indonesia. This study aimed to determine the effects of zinc sulfate (ZnSO4) and synthetic testosterone supplementations for 56 days on testosterone hormone levels and breast muscle performance in Pelung chickens.  This study used 12 Pelung chickens with three treatment groups (G) and four replications, namely, control (G0), ZnSO4 0.9 mg/kg (G1), and synthetic testosterone 3 mg/day (G2). Chickens were acclimatized for 7 days and then supplemented for 56 days. Drinking water and commercial standard feed were provided ad libitum. Blood was collected through the brachial vein for the analysis of testosterone levels using the enzyme-linked immunosorbent assay. The samples were collected every 14 days; on days 0, 14, 28, 42, and 56. Breast muscles were collected for texture analysis, and breast muscle preparations were made with hematoxylin-eosin staining to measure the fascicle area (FA), number of myofibers in one fascicle (NMOF), and myofiber area (MA). The collected data were analyzed using analysis of variance at a 95% confidence level with the help of Statistical Package for the Social Sciences v.29.0. Furthermore, principal component analysis (PCA) was performed with the help of Minitab v. 19.  Statistical analysis results on 56 days of testosterone level parameters showed that G2 was significantly different from all treatments (p < 0.05). The results of statistical analysis on Pelung chicken breast muscle performance, especially hardness, chewiness, FA, NMOF, and MA, were significantly different (p < 0.05) compared with the other treatments. The results of PCA showed that the testosterone level parameters were positively correlated with FA, NMOF, MA, hardness, and chewiness, whereas the fracture parameters were negatively correlated with all parameters except the springiness index and were significantly different between the G2 group and the other groups (p < 0.05).  It can be concluded that supplementing synthetic testosterone 3 mg/day body weight for 56 days can improve testosterone levels and breast muscle performance, especially hardness, chewiness, FA, NMOF, and MA in Pelung chickens. 
Research Article | 27 Oct 2024
Comparative intrafollicular and plasma iron, ferritin, and transferrin concentrations in cycling mares
Katiuska Satué, Esterina Fazio, Gemma Velasco-Martinez, Cristina Cravana, Deborah La Fauci, and Pietro Medica

Volume-17 | Issue-10 | Article-20 | https://doi.org/10.14202/vetworld.2024.2370-2375

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In females of various species and experimental animals, iron (Fe) status in follicular fluid (FF) is associated with local physiological reproductive events related to follicle development, steroidogenesis, and oocyte maturation. However, these mechanisms remain unknown. This study aimed to determine and compare the intrafollicular and plasma concentrations of Fe, ferritin (Ferr), and transferrin (TRF) in cycling mares.  Sixty ovaries were collected during the breeding season from 30 clinically normal mares raised for slaughterhouse meat production. Blood samples were collected before slaughter. Follicles were classified into three categories according to size: Small (20–30 mm; n = 20), medium (≥31–40 mm; n = 20), and large (≥41 mm; n = 20). The FF samples, after collection, were immediately taken to the laboratory for processing and were centrifuged, and the Fe and Ferr concentrations in the supernatant and plasma were determined by spectrophotometry.  Although intrafollicular Fe and Ferr were similar to plasma, TRF was significantly higher in FF than in systemic circulation (p < 0.05). Follicular development does not modify the status of Fe in the mare.  Based on this evidence, it is possible that the acquisition of this molecule possibly originated from a local de novo source, whereas their diffusion through ultrafiltration does not play a relevant role. These results provide new scientific insights into the status of follicle Fe, suggesting its involvement in normal ovarian functions in mares. 
Research Article | 31 Oct 2024
Microbiological profile and prevalence of histamine-producing bacteria in fresh sardines stored at different temperatures
Rachid Khatouf, Said Dahani, Oleya El Hariri, Rajaa Amiyare, and Nourredine Bouchriti

Volume-17 | Issue-10 | Article-21 | https://doi.org/10.14202/vetworld.2024.2376-2384

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The European pilchard (Sardina pilchardus) is an important fish species for the Moroccan economy in terms of production and export. Biogenic amine histamine is a metabolite produced in the flesh of some fish species after death due to the decarboxylation of free histidine by histaminogenic bacteria. Failure to control the histamine risk in European pilchard may lead to public health and socioeconomic issues. This study aimed to estimate the prevalence of histaminogenic bacteria in association with histamine levels and the growth of microflora in Moroccan sardines (European pilchard).  We conducted the study by monitoring Moroccan sardines of histamine content and microbiological profile (aerobic plate count [APC], coliforms, and thermo-tolerant coliforms [TTC]) during 6 days of storage at three different temperatures (0<sup>°</sup>C, 10<sup>°</sup>C, and ambient temperature [22<sup>°</sup>C]). The histamine assay was performed using a spectrofluorometric method, and the microbiological identification of histamine-producing bacteria was performed using a combination of biochemical and molecular tests.  The histamine content in European pilchard stored at 0<sup>°</sup>C was negligible. However, high concentrations were observed at 10<sup>°</sup>C and 22<sup>°</sup>C. The microbiological profile showed a positive association between microflora counts and histamine content according to storage time. At 0<sup>°</sup>C, a moderate increase in the APC, a decrease in coliforms, and an absence of TTC were observed. The rapid proliferation of all microflora was observed at 10<sup>°</sup>C, whereas at 22<sup>°</sup>C, the proliferation was almost exponential. Bacterial identification revealed the exclusive presence of species belonging to the Enterobacteriaceae family at varying frequencies depending on storage temperature. Morganella morganii and Proteus mirabilis had the highest histamine induction rates in L-histidine-supplemented broth, with 1600 and 255 parts per million (ppm), respectively, after 48-h incubation at 35<sup>°</sup>C. Klebsiella ozaenae could produce 136 ppm and Serratia plymuthica 115 ppm. Reverse transcription polymerase chain reaction showed positive results for the presence of genes associated with histidine decarboxylase. The hdc genes of M. morganii, P. mirabilis, and K. ozaenae were successfully amplified and exhibited strong similarity with the reference gene of M. morganii.  This study describes for the first time the hdc gene in bacteria that form histamine in Moroccan sardines. The results also confirm that respect for the cold chain integrity is a crucial factor in histamine management. This information should help stakeholders in the implementation of sound strategies for managing the hazards associated with seafood and their products. 
Research Article | 31 Oct 2024
Digital technologies in dairy cattle breeding to improve the reproductive function of cows and heifers: A case study in Northern Kazakhstan
Rashit Uskenov, Saltanat Issabekova, Aizada Mukhanbetkaliyeva, Orken Akibekov, and Fariza Zhagipar

Volume-17 | Issue-10 | Article-22 | https://doi.org/10.14202/vetworld.2024.2385-2397

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In some countries, the application of digital technologies in dairy cattle breeding is still under development. This study aimed to investigate the use of digital technologies in dairy cattle breeding to improve the reproductive function of cows and heifers in three northern regions of Kazakhstan.  This study explores the application of Austrian smaXtec bolus sensors, which enable the daily monitoring of the reproductive functions of cows and heifers in livestock. To control indicators of reproductive function in Simmental and Holstein-Friesian cattle breeds, a series of experiments were conducted before and after the introduction of boluses in the rumen.  It was established that the application of smaXtec boluses increases milk yield in 305 days, the percentage of conception in the first insemination and in cows with up to three inseminations, the duration of dry secretion, and the percentage of calve output per 100 heads. Moreover, the use of smaXtec boluses reduced the insemination index, duration of the calving-to-conception interval (open days), reproductive rate, and percentage of abortions and culls due to gynecological problems.  The use of smaXtec boluses allows farmers and veterinarians to determine indicators, such as the period of sexual heat in livestock and diseases, in a timely manner and to increase the efficiency of feeding and controlling drinking cycles. Moreover, the application of smaXtec boluses minimizes labor costs associated with collecting data on indicators of reproductive function in cows and heifers and increases accuracy. 
Research Article | 31 Oct 2024
Effects of diluted seawater in drinking water on physiological responses, feeding, drinking patterns, and water balance in crossbred dairy goats
Thiet Nguyen, Khang Van Truong, Khang Kim Thi Nguyen, Ngu Trong Nguyen, Narongsak Chaiyabutr, Narongsak Chaiyabutr, and Sumpun Thammacharoen

Volume-17 | Issue-10 | Article-23 | https://doi.org/10.14202/vetworld.2024.2398-2406

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In tropical regions, the intrusion of saline from seawater (SW) due to global warming and sea level rise in recent years is an important natural factor influencing goat well-being. This study aimed to determine the effects of diluted SW in drinking water on the physiological responses and eating and drinking patterns of crossbred dairy goats under tropical conditions.  Twenty dairy goats were divided into four groups (five animals each) based on body weight and milk yield. Animals received either fresh drinking water (SW0.0, control) or diluted SW at concentrations of 0.5% (SW0.5, low salinity), 1% (SW1.0, moderate salinity), and 1.5% (SW1.5, high salinity). The experiment was performed for 49 days (1st–7th week). Throughout this period, daily food and water intake were measured every day. In addition, blood collection was performed on day 25. Total urine and feces were collected from days 25 to 29. Meal and drinking patterns were determined on days 31 and 32.  Salinity did not influence dry matter intake throughout the experiment (p > 0.05). However, SW had a significant effect on eating patterns. The effect of SW on water intake (WI) was pronounced from the 2nd to 7th weeks of this experiment (p < 0.05). The water balance decreased and plasma antidiuretic hormone levels increased from SW1.5 to SW2.5 compared to the other treatments. Rectal temperature and respiration rate increased from 15:00 to 17:00 in SW1.5 patients. The concentrations of plasma electrolyte, creatinine, and heat shock protein 70 did not differ between treatments (p > 0.05). The urinary excretion of Na+ from SW1.5 and K+ and Cl- from SW1.0 was higher than that from SW0.0 and SW0.5 (p < 0.01).  Lactating crossbred goats adapted to low and moderate SW by increasing urine volume and urinary electrolyte excretion (Uex), whereas animals responded to high SW by either increasing Uex or altering drinking patterns to minimize salt stress. 
Research Article | 31 Oct 2024
Evaluation of galectin-3 and titin in cats with a sarcomeric gene mutation associated with echocardiography
Kanokwan Demeekul, Pratch Sukumolanan, and Soontaree Petchdee

Volume-17 | Issue-10 | Article-24 | https://doi.org/10.14202/vetworld.2024.2407-2416

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Cardiac biomarkers, such as serum galectin-3 (Gal-3) and titin levels, may be related to cats with sarcomeric gene mutations. This study evaluated cardiac biomarkers and echocardiographic parameters in cats with or without myosin-binding protein C3 (MYBPC3) gene mutations.  Forty-two healthy cats without cardiac symptoms, including Bengal, Maine Coon, Scottish fold, and Ragdoll cats, were enrolled in this study. Cats were categorized into three groups: Homozygous wild type (n = 17), homozygous MYBPC3 gene mutation (n = 14), and heterozygous MYBPC3 gene mutation (n = 11). All recruited cats underwent echocardiography, and blood samples were collected for DNA extraction. DNA sequencing for MYBPC3 gene mutations at A31P and A74T loci was first examined by Sanger sequencing. The biomarkers of cardiac fibrosis (Gal-3) and myocardial stiffness (titin) were measured by enzyme-linked immunosorbent assay.  Gal-3 levels >250 pg/mL were associated with echocardiographic parameters. However, Gal-3 levels were not significantly different between cats with MYBPC3 gene mutations and those in the wild-type group. Titin was associated with the left ventricular (LV) thickness and systolic function (r = 0.405, p = 0.013). Qualitative measurement of titin antibodies showed that the highest percentage of these antibodies was found in homozygous wild-type cats. No correlation was found between titin levels and MYBPC3 gene mutations. Weight was positively associated with interventricular septum (r = 0.312, p = 0.056) and LV wall thickness (LVPW) (r = 0.219, p = 0.187). However, they were not associated with Gal-3 levels.  LVPW was correlated with weight in cats with sarcomeric gene mutations. Serum titin may be an underlying factor for cardiac hypertrophy in cats.