Serological and epidemiological investigation of Mycobacterium avium subspecies paratuberculosis in bovines in Pakistan.

Objective: To investigate the prevalence of paratuberculosis in cattle and buffaloes at twelve public dairy farms in Punjab, Pakistan. Methods: A total of 2181 more than two-year-old animals (1242 cattle and 939 buffaloes) were tested by avian tuberculin, i.e., killed purified protein derivative (PPD) of Mycobacterium avium paratuberculosis and indirect ELISA. Blood and fecal samples were collected from tuberculin positive animals. These samples were further processed by indirect ELISA. The data were analyzed using frequency analysis and logistic analysis procedures. Results: The prevalence of paratuberculosis at public dairy farms was 3.8%, as determined by tuberculin + ELISA test. It varied from 0.71-13.5% with a 100% herd prevalence. Multivariate logistic regression analysis revealed that species, milk production, total animals, total small ruminants, and total buffaloes were significantly associated with the occurrence of paratuberculosis. Odd ratio analysis revealed that with a one-kilogram increase in body weight, there will be a 0.006% increase in disease occurrence. With the increase in one animal in small ruminants and buffaloes, there will be 0.008% and 0.42% greater chances of developing paratuberculosis, respectively. Bivariate logistic regression analysis of cattle and buffaloes revealed that farm number, age, and total number of cattle were significantly associated with the occurrence of paratuberculosis. A one-month increase in lactation length increases the chance of tuberculosis by 0.004%; similarly, a one-liter increase in milk production increases the chance of disease by 10%. With each additional buffalo in the herd, there will be a 0.007% greater chance for the occurrence of paratuberculosis. Conclusion: This study concluded that tuberculin testing can be used in conjunction with ELISA to screen animals for paratuberculosis in countries with scarce resources, such as Pakistan. The efficacy of disease diagnosis can be improved by combining multiple tests.

Unlocking the anti-diabetic potential of Gymnema sylvestre, Trigonella foenum-graecum, and their combination thereof: An in-vivo evaluation.

The current study aimed to explore the anti-diabetic effect of aqueous extracts of Gymnema sylvestre, Trigonella foenum-graecum and mixture of both the plants in alloxan-induced diabetic rabbits. A total of 30 rabbits were grouped into six equal groups as: normal control, diabetic control, diabetic treated with 300 mg/kg body weight (bw) G. sylvestre, diabetic treated with 300 mg/kg bw T. foenum-graecum, diabetic treated with 300 mg/kg bw mixture of both the plants and diabetic treated with 500 mg/kg bw metformin for 4 weeks. Diabetes was induced to all the study group animals except normal control by intravenous administration of alloxan monohydrate (80 mg/kg bw). Blood glucose was measured by glucometer and other biochemical parameters were determined through various kit methods. Serum insulin was measured through ELISA kit method. Results showed that both the plants and metformin significantly (p < .05) decreased the fasting blood glucose. Hypoglycemic activity of aqueous extract of G. sylvestre and metformin was found slightly higher than aqueous extract of T. foenum-graecum and the mixture of both the plants. However, a significant (p < .05) rise in insulin secretion was observed in studied plants extract treated rabbits. Serum urea, creatinine, and liver enzymes were found reduced significantly (p < .05) in treated rabbits whereas packed cell volume was also returned to normal in treated animals as compared to control group. The study concluded that G. sylvestre and T. foenum-graecum extracts have comparable effects with metformin in normalizing the blood glucose level and have more pronounced effect than metformin in restoring the serum biochemical parameters to normal levels. Hence, these plants may be the good alternative medicine in managing the diabetes mellitus.

Seroprevalence and haemato-biochemical effects of bovine leucosis in buffalo, Punjab, Pakistan.

Enzootic bovine leucosis is caused by bovine leukaemia virus (BLV), a Deltaretrovirus belonging to the family Retroviridae. BLV causes huge economic losses to the dairy industry in the form of decreased milk production, premature culling, and poor reproductive performance of the animals. The aim of the present study was to determine the seroprevalence of BLV infection in buffalo in two districts of Punjab, Pakistan. A total of 384 samples were collected and analysed using a commercial indirect enzyme-linked immunosorbent assay (ELISA) to investigate the seroprevalence of BLV through the detection of the anti-BLV gp51 antibody. A predesigned data questionnaire proforma was employed to find out the association of risk factors with disease. Overall, 18.2% of buffaloes were seropositive for BLV in the study population. The results revealed a significant association (P < 0.05) of age with BLV infection. Furthermore, milk yield and pregnancy had a significant association with the seroprevalence of BLV infection in buffalo whereas no significant association was found with sex, breeding, and health status. Biochemical and oxidative stress markers revealed a significant decrease in liver enzymes alanine transaminase (ALT) and aspartate transaminase (AST), glutathione peroxidase (GPX), and superoxide dismutase (SOD) in seropositive animals as compared to healthy animals. It is concluded that BLV has a considerable prevalence in buffalo in Punjab, Pakistan and there is a dire need to investigate the disease epidemiology at both national and international levels and strategies should be developed to implement an effective control program.

Incidence of aflatoxin M1 in cows' milk in Pakistan, effects on milk quality and evaluation of therapeutic management in dairy animals.

The present study was aimed at measuring the concentration of aflatoxin M1 (AFM1) in the milk of Holstein Friesian cows, its effect on the milk quality and seasonal trends, as well as to investigate the efficacy of a commercial clay-based toxin binder. For this purpose, milk samples from dairy cows (n = 72) were collected and assayed for AFM1 before employing a clay-based toxin binder. The milk samples (n = 72) were collected from selected animals, revealing that 69.4% of the milk samples had AFM1 levels above the United States permissible limit (0.5 µg/kg). The incidence of AFM1 in milk during the winter and summer was 82.5% and 53.1%, respectively. Owing to the presence of AFM1, the level of milk fat, solids-not-fat, and protein were found to be low. Subsequently, the affected animals were divided into two groups, i.e., AFM1 positive control (n = 10) and the experimental group (n = 40). The experimental group of animals were fed the clay-based toxin binder at 25 g/animal/day. A progressive decrease of 19.8% in the AFM1 levels was observed on day 4 and on day 7 (53.6%) in the treatment group. Furthermore, the fat, solids-non-fat and protein increased significantly in the milk. In conclusion, a high level of AFM1 contamination occurs in the milk in Pakistan, affecting the quality of the milk production. Clay-based toxin binders may be used to ensure the milk quality and to protect the animal and consumer health.

Corrigendum: Sero-epidemiological study of zoonotic bacterial abortifacient agents in small ruminants.

[This corrects the article DOI: 10.3389/fvets.2023.1195274.].

Detection and Phylogenetic Analysis of Extended-Spectrum ß-Lactamase (ESBL)-Genetic Determinants in Gram-Negative Fecal-Microbiota of Wild Birds and Chicken Originated at Trimmu Barrage.

Extended-spectrum ß-lactamases (ESBL) give rise to resistance against penicillin and cephalosporin antibiotics in multiple bacterial species. The present study was conducted to map genetic determinants and related attributes of ESBL-producing bacteria in three wild aquatic bird species and chickens at the "Trimmu Barrage" in district Jhang, Punjab province, Pakistan. To study the prevalence of ESBL-producing bacteria, a total of 280 representative samples were collected from wild bird species; cattle egrets (Bubulcus ibis), little egrets (Egretta garzetta) and common teals (Anas crecca) as well as from indigenous chickens (Gallus gallus domesticus) originating from a local wet market. The isolates were confirmed as ESBL producers using a double disc synergy test (DDST) and bacterial species were identified using API-20E and 20NE strips. A polymerase chain reaction (PCR) was used to detect ESBL genetic determinants and for genus identification via 16S rRNA gene amplification. A phenotypic antimicrobial susceptibility test was performed for ESBL-producing isolates against 12 clinically relevant antibiotics using the Kirby-Bauer disk diffusion susceptibility test. A phylogenetic tree was constructed for the sequence data obtained in this study and comparative sequence data obtained from GenBank. The overall prevalence of ESBL-producing bacteria was 34.64% (97/280). The highest percentage (44.28%; 31/70) of ESBL-producing bacteria was recovered from chickens (Gallus gallus domesticus), followed by little egrets (Egretta garzetta) (41.43%; 29/70), common teal (Anas crecca) (28.57%; 20/70) and cattle egrets (Bubulcus ibis) (24.28%; 17/70). Five different ESBL-producing bacteria were identified biochemically and confirmed via 16S rRNA gene sequencing, which included Escherichia coli (72; 74.23%), Enterobacter cloacae (11; 11.34%), Klebsiella pneumoniae (8; 8.25%), Salmonella enterica (4; 4.12%) and Pseudomonas aeruginosa (2; 2.06%). Based on PCR, the frequency of obtained ESBL genes in 97 isolates was blaCTX-M (51.55%), blaTEM (20.62%), blaOXA (6.18%) and blaSHV (2.06%). In addition, gene combinations blaCTX-M + blaTEM, blaTEM + blaOXA and blaCTX-M + blaSHV were also detected in 16.49%, 2.06% and 1.03% of isolates, respectively. The ESBL gene variation was significant (p = 0.02) in different bacterial species while non-significant in relation to different bird species (p = 0.85). Phylogenetic analysis of amino acid sequence data confirmed the existence of CTX-M-15 and TEM betalactamases. The average susceptibility of the antibiotics panel used was lowest for both Klebsiella pneumoniae (62.5% ± 24.42) and Salmonella enterica (62.5% ± 31.08) as compared to Enterobacter cloacae (65.90% ± 21.62), Pseudomonas aeruginosa (70.83% ± 33.42) and Escherichia coli (73.83% ± 26.19). This study provides insight into the role of aquatic wild birds as reservoirs of ESBL-producing bacteria at Trimmu Barrage, Punjab, Pakistan. Hence, active bio-surveillance and environment preservation actions are necessitated to curb antimicrobial resistance.

Sero-epidemiological study of zoonotic bacterial abortifacient agents in small ruminants.

Abortion is one of the leading causes of economic losses in the livestock industry worldwide. Chlamydia abortus, Coxiella burnetii, and Brucella spp. are the leading cause of late fetal loss in small ruminants. This study determined the seroprevalence of these agents in small ruminants in district Jhang. A total of 385 serum samples were taken from the sheep and goats from different flocks with a history of abortion and subjected to i-ELISA. Further, samples were analysed for liver enzymes and total serum protein using a semi-automated chemistry analyzer. The result of indirect commercial ELISA showed 13.0, 4.2 and 11.2% prevalence for Coxiella burnetii, Chlamydia abortus, and Brucella spp., respectively. Values of different serum parameters (ALT, AST, and total protein) of seropositive animals were also determined. There was a significant rise in AST and ALT values of infected animals (p ≤ 0.05). Total protein decreased for all three infections, but a significant drop was noted in Brucella positive sheep serum samples. Various risk factors were studied. Binary logistic regression proved a significant role of ticks for coxiellosis and brucellosis. Age, parity, and species did not impact the prevalence of diseases studied.

Genomic Diversity and Evolution of SARS-CoV-2 Lineages in Pakistan.

The emergence of SARS-CoV-2 variants has posed a challenge to disease control efforts worldwide. This study explored the genomic diversity and phylogenetic relationship of SARS-CoV-2 variants reported in Pakistan. Our objective was to understand the transmission dynamics of different lineages within the country. We retrieved and analyzed spike protein sequences from Pakistan and compared them with reference sequences reported worldwide. Our analysis revealed the clustering of Pakistan-origin isolates in nine different clades representing different regions worldwide, suggesting the transmission of multiple lineages within the country. We found 96 PANGO lineages of SARS-CoV-2 in Pakistan, and 64 of these corresponded to 4 WHO-designated variants: Alpha, Beta, Delta, and Omicron. The most dominant variants in Pakistan were Alpha (B.1.1.7), Beta (B.1.351), Delta (B.1.617.2, AY.108), and Omicron (BA.2.75, BA.5.2), and the N-terminal domain and receptor binding regions were the most hypervariable regions of the spike gene. Compared to the reference strain, characteristic substitutions were found in dominant variants. Our findings emphasize the importance of continuously monitoring and assessing nucleotide and residue substitutions over time to understand virus evolutionary trends better and devise effective disease control interventions.

Serological, molecular detection and potential risk factors associated with camel brucellosis in Pakistan.

Brucellosis is one of the most important zoonoses in developing countries and was considered the most widespread zoonosis in the world. Brucellosis was reported in camels and has been reported from all camel-keeping countries.The present study was performed in three districts (Jhang, Chiniot, and Bhakkar) of Punjab province of Pakistan. A total of 200 camel (Camelus bactrianus) sera were collected using random and multistage cluster sampling from different areas. Fifty samples were collected from one organized governmental farm. One hundred fifty samples were collected randomly from nomadic/pastoral production systems. All sera were tested with Rose Bengal plate agglutination test (RBPT) and confirmed by ELISA. Genomic DNA was extracted from all serum samples and tested by real-time PCR. Various potential risk factors (season, rearing with other animals, and abortion or orchitis history) recorded through questionnaires were statistically analyzed by Chi-square test.In total, 5 % of investigated sera were positive by RBPT. Only 2 % of the camel sera were CELISA positive. Brucella abortus DNA was detected in 1.5 % of the investigated animals. Season, rearing of camels with other ruminants, abortion, and orchitis history were found to be statistically significant (p < 0.05) disease for determinants.Camel brucellosis is a zoonotic disease in the Pakistani Punjab with various risk factors maintaining and perpetuating its spread. Therefore, there is a need for implementing control measures and raising public health awareness in prevention of brucellosis in Pakistan.

Evaluation of furazolidone, sulfadimidine and amprolium to treat coccidiosis in Beetal goats under field conditions.

Coccidiosis is a protozoal and occasionally fatal diarrheic disease of goats imposing heavy economic losses to farming community. This study aimed to evaluate the efficacies of Furazolidone, Sulfadimidine and Amprolium against coccidiosis in Beetal goats. Twenty-four (24) Beetal goats naturally infected with coccidiosis were randomly divided into four groups of 6 (A-D). Goats in groups A, B and C were treated orally with Furazolidone (10 mg/Kg), Sulfadimidine (100 mg/Kg) and Amprolium (55 mg/Kg), respectively for 7 days. Goats in-group D served as positive control. Oocysts per gram (OPG) of feces counts of individual goats in each group were performed on Days; 0 (pre-treatment) 7, 14 and 21 (post-treatment). OPG counts amongst goats in all groups at day 0 were not significant (P>0.05). On days 7, 14 and 21, OPG values decreased significantly (P<0.05) in groups A, B and C compared to group D. The efficacy of Furazolidone, Sulfadimidine and Amprolium was 98.6, 98.0 and 99.6 percent, respectively on Day 21 (end of trial). Statistically, the efficacies of three drugs were not significantly different (P>0.05). In conclusion, Furazolidone, Sulfadimidine and Amprolium are well-tolerated and any one of these may be recommended to effectively treat coccidiosis in Beetal goats.

A review of Neospora caninum in water buffalo (Bubalus bubalis).

A number of countries in the world have reported infections with Neospora caninum in water buffalo (Bubalus bubalis), from Africa to Asia, Europe and South America and recently Australia. In general, clinical manifestations (such as abortion) seem rare, which has raised the prospect that buffalo may be inherently resistant to clinical effects of N. caninum infection. Worldwide, the seroprevalence of N. caninum infection (as a measure of exposure determined by the detection of antibody) in buffalo is high, at approximately 48%. This reported seroprevalence is three or four times higher than that reported from the world's cattle populations, which have collective seroprevalence rates of 16.1% for dairy cattle and 11.5% for beef cattle. However, there is a lack of standardisation in seroprevalence studies and some studies may well under-estimate the true level of infection. Epidemiologic evidence supports post-natal transmission, and in utero transmission has also been demonstrated. The causes for water buffalo to have markedly higher seroprevalence but apparently lower neosporosis abortion rates than cattle warrant further investigation.

Prevalence and distribution of Neospora caninum in water buffalo (Bubalus bubalis) and cattle in the Northern Territory of Australia.

The seroprevalence of Neospora caninum infection in water buffalo (Bubalus bubalis) and domestic cattle in the Northern Territory (NT) of Australia has never been determined. A total of 480 serum samples from water buffalo and 192 serum samples from cattle, collected by the NT Government from 1993 through to 2001, at 18 different survey sites throughout the Northern Territory were tested by commercial ELISA for anti-N. caninum antibodies. The water buffalo samples demonstrated a seroprevalence of 88.3% (95% CI ± 2.9%), while 31.8% (±6.1%) of the cattle sera tested positive for N. caninum antibodies. Individual buffalo from the same herd, sampled over years, showed considerable fluctuations in S/P ratios. Overall, seropositivity was consistent across buffalo herds, and showed a slight decline over the years. The study presents evidence for the first time that N. caninum infection in water buffalo in the Northern Territory is a highly endemic and that infection rates are higher than those for cattle. This is important for an understanding of any potential sylvatic life cycle of N. caninum in Northern Australia. This survey also tests cattle from that territory for the first time for evidence of N. caninum infection and makes an important contribution to the understanding of disease management issues for the beef industry in the region.

The effect of infection with mixed Eimeria species on hematology and immune responses following Newcastle disease and infectious bursal disease booster vaccination in broilers.

BACKGROUND: Coccidiosis is an important parasitic disease of chickens, causing high mortality and morbidity. This morbidity is believed to be correlated with altered population dynamics of blood cells and immunocompromisation. OBJECTIVES: This study investigated the effects of mixed Eimeria species (viz., tenella, maxima, acervulina and necatrix) infection on hematology and immune responses following Newcastle disease (ND) and infectious bursal disease (IBD) booster vaccination in broilers. ANIMALS AND METHODS: One-day-old broiler chicks (Hubbard; n = 200) were divided into two equal groups A and B. On day 16, group A was infected orally with Eimeria species (7 × 10(4) sporulated oocysts), whereas group B served as control. Both groups were analyzed for hematological parameters on post-infection days 6-8. Sera from both groups were analyzed for antibody titers against ND and IBD vaccines. On day 8 post-infection, lymphoid organs were also examined. RESULTS: Significantly lower (P < 0.05) levels of plasma proteins, globular volume, hemoglobin concentration, packed cell volume, total erythrocytes, mean corpuscular volume and mean corpuscular hemoglobin concentration were found in infected chickens compared with non-infected control chickens. In addition, the infected group exhibited significantly increased (P < 0.05) numbers of different leukocytes. Infected chickens also showed significantly lower antibody titers against ND and IBD with decreased relative organ weights of all lymphoid organs except spleen. CONCLUSION AND RECOMMENDATIONS: Mixed species of Eimeria adversely affected the hematology and immune efficiency of broilers. Thus, inexpensive immune potentiators and hemotonics along with appropriate anti-coccidial medications are suggested to avoid the complications and subsequent economic losses.

Effect of oxytocin on serum biochemistry, liver enzymes, and metabolic hormones in lactating Nili Ravi buffaloes.

Studies reporting the effects of oxytocin on the health of lactating animals are lacking and still no such data is available on Nili Ravi buffalo, the most prominent Asian buffalo breed. The present study was conducted to investigate the effect of oxytocin on physiological and metabolic parameters of lactating Nili Ravi buffaloes. Healthy lactating buffaloes (n = 40) of recent calving were selected from a commercial dairy farm situated in the peri-urban area of district Faisalabad, Pakistan. These buffaloes were randomly allocated to two equal groups viz experimental and control, comprising 20 animals each. Twice-a-day (morning and evening) milking practice was followed. The experimental and control buffaloes were administered subcutaneously with 3 mL of oxytocin (10 IU/mL) and normal saline respectively, prior to each milking. Serum biochemical profile including glucose, total cholesterol (tChol), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), triglycerides (TG), total proteins (TP), C-reactive protein (CRP), liver enzymes aspartate transaminase (AST), alanine transaminase (ALT), and metabolic hormones triiodothyronine (T3) and thyroxine (T4) were studied. Results revealed significantly higher (P ≤ 0.01) levels of glucose, total cholesterol, LDL-C, triglycerides, total proteins, and C-reactive protein in experimental (oxytocin-injected) lactating buffaloes compared to control group. Liver enzymes AST and ALT as well as serum T4 concentration was significantly higher (P ≤ 0.01) in oxytocin-injected lactating buffaloes as compared to control animals. It was concluded that oxytocin had the key role in increasing the metabolic parameters and hormones, resulting in the optimization of production. But, at the same time, it may pose a threat to the animal health.

Some facts and issues related to livestock theft in Punjab province of Pakistan --findings of series of cases.

Stock theft is an endemic crime particularly affecting deep rural areas of Pakistan. Analysis of a series of cases was conducted to describe features of herds and farmers who have been the victims of cattle and/buffalo theft in various villages of Punjab in Pakistan during the year 2012. A structured interview was administered to a sample of fifty three affected farmers. The following were the important findings: i) incidents of theft were more amongst small scale farmers, ii) the rate of repeat victimization was high, iii) stealing was the most common modus operandi, iv) the majority of animals were adult, having high sale values, v) more cases occurred during nights with crescent moon, vi) only a proportion of victims stated to have the incident reported to the police, vii) many farmers had a history of making compensation agreements with thieves, viii) foot tracking failed in the majority of the cases, ix) all the respondents were willing to invest in radio frequency identification devices and advocated revision of existing laws. The study has implications for policy makers and proposes a relationship between crime science and veterinary medicine.

Sero-prevalence of Neospora caninum and Besnoitia besnoiti in South Australian beef and dairy cattle.

A serological survey for Neospora caninum and Besnoitia besnoiti was carried out in beef and dairy cattle in South Australia. Serum samples of dairy cattle (n=133) from 9 properties and tank milk samples from a further 122 dairy herds were tested. An additional 810 sera from beef cattle from 51 properties were also tested. Testing at the individual animal level by IDEXX NEOSPORA X2 Ab test ELISA revealed a low prevalence of N. caninum antibodies of only 2.7% (95% CI; 1.6-3.7%) sera positive, as did the milk testing that showed 2.5% (95% CI; 1.4-3.6%) of tank milks being positive. At the herd level, 29.4% (95% CI; 16.9-41.9%) of beef, and 44.4% (95% CI; 12.0-76.9%) of dairy cattle herds showed serum antibodies. The highest within-herd prevalence in beef was 20% and 25%in dairy, which explains the low herd prevalence in dairy detected by bulk milk testing. Testing for B. besnoiti antibodies by PrioCHECK(®) Besnoitia Ab 2.0 ELISA initially identified 18.4% (95% CI: 15.8-21.0%) of 869 individual cattle sera as positive by ELISA at the manufacturer's suggested cut-off threshold (15 PP). Additional tests by immunoblot and IFAT, however, could not confirm any of the ELISA results. The use of a higher (40 PP) threshold in the ELISA is suggested to improve specificity. There is thus no evidence of B. besnoiti infection in South Australian cattle.

Modified picrate method for determination of cyanide in blood.

Cyanide (CN-) is widely distributed in the ecosystem and has been associated with toxic effects in humans and animals. Most outbreaks of CNÖ¿ poisoning in animals result from ingestion of plants containing cyanogenic glycosides. Various analytical techniques for estimating cyanide in blood are available. A simple picrate method was developed to determine blood CN- in goats. This assay is a modification of commonly available methods using picrate paper and those using Conway diffusion cells. Cyanide in blood was measured during and after IV administration of KCN at 0.6 mg/min for 1 h. Blood CN- levels in rabbits were determined after oral administration of KCN for 10, 20, 30 and 40 days. The CN- concentration in blood of goats was time-dependent and continued rising during infusion followed by gradual decline after infusion stopped. A calibration curve set by dissolving various concentrations of KCN in distilled water showed a linear relationship between CN- concentration and absorbance (R=0.995) ranging from 0.3-120 mg CN-/L. Blood CN- levels in rabbits showed time-dependent increase with maximum concentration (1.34 mg/L) at 40 days. This is a simple and inexpensive tool for the determination of blood CN- in the laboratory and under field conditions as well.

Exposure of smoke solutions from CNG-powered four-stroke auto-rickshaws induces distressed embryonic movements, embryonic hemorrhaging and ectopia cordis.

In south Asian countries, a campaign has launched to promote CNG-powered four-stroke auto-rickshaws (CNFAR) to decrease emission load in the environment. Even though, CNFAR are considered environmentally safe, emissions of some other toxic chemicals would amplify, which may effect the development of growing fetus and may result in different growth defects. By utilizing the in vivo chicken embryo model, this report analyzes the toxic potential of CNFAR smoke solutions (CNFARSS) on embryonic movements (EM) and cardiovascular development. Application of CNFARSS to embryos caused profound decline (p<0.001) in all four types of EMs. Several recovery attempts of all EMs were observed in oscillating fashion, however, EMs did not recover by the end of experiment. Exposure of CNFARSS escorted intense decline (p<0.001) with temperate recovery phases in the EM of tail. Macroscopic evaluation of all CNFARSS treated chicken embryos revealed several widespread hemorrhaging throughout the whole body. Moreover, four different types of ectopia cordis were prominently observed among all CNFARSS treated embryos, namely; incomplete ectopia cordis, complete ectopia cordis, cervico-thoracic ectopia cordis and thoraco-abdominal ectopia cordis.